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POSTERS p = 0.008). MC displayed a highly enriched Vdelta1 pool compared to HC and CHC (2.9% vs 1.0% and 1.1% of circulating CD3, p = 0.008 for both) that determined the inversion of the Vdelta1/Vdelta2 ratio (5.9 [MC] vs 0.5 [HC] and 0.6 [CHC], p = 0.003 for both). This ratio inversely correlated to cryocrit percentage (R2 = 0.39, p = 0.05). Conclusion: The reduced ability of MC T-regs to inhibit IFN-gamma production by target cells and the correlation between CD4+CD25− proliferation in co-cultures and cryocrit percentage suggest a role for T-reg impairment in perpetuating mixed cryoglobulinemia, whereas a predominance of Vdelta1 among gamma-delta T cell subsets may be important to limit cryocrit formation. 699 PROTEIN KINASE C b PLAYS A VITAL ROLE IN STAT1 AND STAT3 ACTIVATION IN HEPATOCYTES IN RESPONSE TO INTERFERON-a S. Mullins1,2 , S. Norris2 , A. Long1 . 1 Institute of Molecular Medicine, Trinity Centre for Health Sciences, St James’s Hospital, 2 Hepatology, St James’s Hospital, Dublin, Ireland E-mail: [email protected] Background and Aims: At present, combination therapy with interferon-a and ribavirin remains the cornerstone of the treatment of Hepatitis C virus (HCV). With response rates to interferon as low as 42% in patients with genotype 1, the mechanism of interferon resistance remains elusive. It has been demonstrated that classical protein kinase C isoforms (PKC-a, -b, -g) are involved in signal transducers and activators of transcription (STAT) activation.1 Furthermore, HCV viral proteins have been shown to modulate PKC-b activity in lymphocytes leading to inhibition of cell migration.2 We investigate PKC and interferon signalling via the Janus kinase (Jak)/STAT pathway. Our aim is to identify which cPKC isoform is required for interferon signalling leading to STAT1 and STAT3 activation in hepatocytes. Methods: PKC-a and PKC-b were inhibited in Huh7 cells using pharmacological inhibitors as well as RNA interference, prior to treatment with clinical grade interferon-a. Phosphorylation of STAT1 and STAT3 was then assessed by Western blot, as a measure of Jak/STAT pathway signalling capacity. Results: Interferon-a induces phosphorylation of STAT1 at Tyr701 and STAT3 at Tyr705. The classical PKC inhibitor, Go6976, caused complete attenuation of STAT3 phosphorylation with a reduction in STAT1 phosphorylation. By knocking down PKC-a alone using siRNA, no attenuation of either STAT1 or STAT3 phosphorylation was observed. However, using a PKC-b-specific inhibitor, LY333531, STAT1 activation was reduced while STAT3 phosphorylation was completely absent. This result was confirmed with siRNA knockdown of PKC-b. Conclusions: We demonstrate for the first time that the PKC-b isoform is an essential kinase required for the integrity of the interferon signalling pathway. This result represents an extremely important development in determining the mechanism of interferon resistance in HCV. Reference(s) [1] Fimia G.M., et al. J. Virol, 2004. 78(23): p. 12809–16. [2] Volkov Y., et al. Gastroenterology, 2006. 130: p. 482–492.

700 INFLAMMASOME IS MODULATED BY PEGYLATED INTERFERON MONOTHERAPY IN CHRONIC HEPATITIS C PATIENTS M.G. Neuman1,2 . 1 Pharmacology, In Vitro Drug Safety and Biotechnology, 2 Pharmacology, University of Toronto, Toronto, ON, Canada E-mail: [email protected] Pegylated-interferon (PEG-IFN-a) monotherapy is needed in hepatitis C virus (HCV) infected-patients not-tolerating ribavirin. The aim was to evaluate the inflammasome in HCV patients, to correlate serum apoptosome, interleukins, RANTES, pathogenS272

associated-molecular-pattern (PAMP), plasminogen-activator-inhibitor 1(PAI-1), tumour necrosis factor-alpha (TNFa), and transforming growth factor-beta (TGFb) levels with the severity of HCV, and the responses to PEG-IFNa-2b. Methods: 180-non-cirrhotic patients were part of a randomized, double-blind-dose-finding clinical trial PegIntron™ (ScheringPlough) to study efficacy of 0.5, 1.0 and 1.5 mg/kg/week for 48 weeks. The patients were grouped by PEG-IFN-dose received. Each group was stratified by responses: sustained-response-SR [HCV-RNA undetectable 6 months after the end-of-therapy (ET)], relapse-response-RR (HCV-RNA undetectable ET) or no-responseNR (detectable HCV-RNA at ET). Serum inflammasome-levels were measured by ELISA. Student-t-test with Bonferonni correction determined the significance between the groups. The c2 test or Fisher’s exact test compared the frequency of data between groups. Results: Initially, there were no statistical differences regarding the demographics, viral load, genotypes, apoptosome, inflammasome, histological-activity-index (HAI) and fibrosis scores between groups. Of 180 patients; 3 had 0 HAI, 47-mild, 121-moderate and 9-high; and had Metavir-fibrosis (MF0–5; MF1–152; MF2–13; MF3–10). A good correlation was seen between the HAI and TNF-a levels (r = 0.92, p < 0.001) in all the patients (r = 0.85; p < 0.001). IL-8 and RANTES increased significantly at MHAI-3 versus lower MHAI1–2. TGFb increased significantly with the severity of fibrosis. TNFa and apoptosis were lower at the base-line in SR versus RR and NR. This is the first study to illustrate that in monotherapy there is a correlation between the HAI-reduction, the decrease of TNFa and apoptosis. Regardless the dose of therapy, TNF-a and TGFb decreased significantly in SR-patients versus their initial values. PAMP and PAI-1 did not demonstrate differences between the doses. There was a statistical difference (p < 0.5) between initial levels in SR and NR. Conclusion: Low baseline serum TNFa and apoptosome are predictors for SR. PEG-IFN-a reduces inflammasome contributing to reduce fibrosis. Acknowledgements: Hepatitis Interventional Therapy Group was involved in the clinical trial. We are thankful to Clinical Research Hepatology-GastroIntestinal Schering-Plough Research Institute, that provided access to these samples and clinical data. 701 HOST GENETIC DETERMINANTS IN HCV-RELATED MIXED CRYOGLOBULINEMIA A. Piluso, L. Gragnani, P. Caini, E. Fognani, C. Giannini, M. Monti, A. Petrarca, J. Ranieri, G. Laffi, A.L. Zignego. MASVE Center, Department of Internal Medicine, Universi` a degli Studi di Firenze, Firenze, Italy E-mail: [email protected] HCV infection is strictly related to Mixed Cryoglobulinemia (MC), a lymphoproliferative/autoimmune disorder characterized by circulating immunoglobulin complexes (cryoglobulins). The reasons why only a percentage of HCV patients develops MC are still unknown. Two – not mutually exclusive – hypotheses could be suggested: (i) a reduced uptake/clearance of Igs by phagocytes, (ii) an excessive production/secretion of Igs. The clearance of cryoglobulins is mediated by low-affinity Fcg receptors (FcgRs). Single nucleotide polymorphisms affecting IgG-binding affinity are described for some receptors and particular aplotypes are related to autoimmunity. On the other hand, MC is characterized by B-cell expansion and abnormal Ig production involving specific cytokines, namely BAFF (B-cell Activating Factor). This study was aimed at evaluating the contribution of genetic host factors in the development of HCV-related MC. We analyzed, with different PCR-based techniques and RFLPs, the prevalence of FcgR polymorphisms (FcgR2A-131 R/ A, FCgR2B232I/T, FCgR3A-176V/F and FCgR3B-NA1/NA2) in 210 HCV+ patients

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POSTERS (102 with MC(MC+) and 108 (MC-) without any evidence of lymphoproliferative/autoimmune disorders). The BAFF promoter polymorphism −871C/T, inducing a higher transcription of the BAFF gene, was also analyzed. A significantly higher prevalence of both T/T homozygosis (p < 0.001) and the presence of a T-allele (T/T and T/C, p = 0.004) at site −871 of BAFF promoter was shown in MC+ patients, resulting in a significant increase of BAFF serum concentration (4.12±1.29 vs. 2.09±0.81 ng/ml, p < 0.0001). Real-time-PCR analysis of the BAFF gene expression showed a 2.8-fold higher transcription in MC+. By contrast, no significant difference in prevalence of different FcgRs genotypes was observed. However, a trend towards a higher prevalence of the FCgR3A-F/F genotype – showing a reduced affinity for IgG – was shown in MC+patients (p < 0.09). Interestingly, in 19 MC+patients undergoing anti-CD20 antibody treatment, the response was strictly related to the F-allele presence, being strongly reduced in F/F homozygous patients. These results underline the importance of the host genetic background in the development of HCV-related MC, suggesting that mechanisms enhancing Ig production and B-cell survival have a more relevant role than genetic defects in cryoglobulin clearance. This latter mechanism, however seems to be very important in the effectiveness of biological therapies, frequently used in HCV-related MC. 702 SPONTANEOUS RESOLUTION OF ACUTE HEPATITIS C VIRUS (HCV) INFECTION CORRELATES WITH THE RECONSTITUTION OF THE CIRCULATING CD56DIM NATURAL KILLER (NK)-CELL POOL A. Riva1 , S. Phillips1 , A. Evans1 , A. Ambrozaitis2 , R. Williams1 , N.V. Naoumov1 , S. Chokshi1 . 1 Institute of Hepatology, University College London, London, UK; 2 Department of Infectious Diseases, Vilnius University, Vilnius, Lithuania E-mail: [email protected] Background and Aims: Efforts to identify the immune-correlates responsible for resolution of HCV infection are fundamental to develop new treatment strategies and an effective vaccine against HCV. We have previously shown that imbalanced NK-cell subsets, with hyper-expression of co-inhibitory markers, are associated with chronic HCV infection. In this study we aim to assess the role of NK-cells in determining the outcome of acute HCV infection in a cohort of well characterized patients. Methods: We analysed 12 patients with acute HCV infection who met the following criteria: ALT >10xULN, exposure to HCV within previous 4 months and HCV-RNA(+). Viral load was determined by qPCR. Peripheral blood mononuclear cells collected at 3 timepoints (baseline, BL; month-1, M1; month-6, M6) were stained with fluorochrome-labelled antibodies to NK-cells (CD3/CD56/CD16). Proportions of CD56dim (CD16bright ) and CD56bright (CD16dim ) subsets and expression of PD-1/PD-L1 were evaluated by 6-colour flow cytometry and correlated with HCV-RNA and ALT at each timepoint and over-time. Supernatants from cell-cultures in the presence of HCV-antigens were collected for cytokine analysis and quantification. Results: Six patients resolved HCV spontaneously (Resolvers), whilst 6 developed chronic infection (Chronics). Frequencies of total CD3neg /CD56pos -(NK)-cells were similar in Resolvers and Chronics. However, proportions of CD56dim and CD56bright NK subsets differed. In Resolvers, CD56dim cells became progressively predominant over CD56bright , and only in Resolvers did the CD56dim /CD56bright -ratio increase over-time (BL-to-M1, p = 0.014; M1-to-M6, p = 0.030); at M6 it was higher than in Chronics(p = 0.048). PD-L1 expression on CD56dim was always lower in Resolvers (p ≤ 0.02). Analysis of clinical parameters revealed that in Resolvers the progressive increase of CD56dim /CD56bright -ratio correlated with the decline of HCV-RNA over-time (r = −0.997, p = 0.042), due to reduction of

CD56bright (r = 0.999, p = 0.011) and expansion of CD56dim (r = −0.997, p = 0.041). At M1, Resolvers’ CD56dim were positively correlated with ALT (r = 0.820, p = 0.046). Conclusions: In patients with acute HCV infection the proportions and evolution of the two functionally distinct NK-cell subsets differ in patients that subsequently resolve infection compared to those who become chronically infected. A favourable outcome of infection is associated with the establishment of a defined NK profile with predominance of CD56dim (cytotoxic) NK-cells, which may be due to the clearance of virus-infected hepatocytes as shown by the correlation of this subset with serum HCV-RNA decline and ALT levels. 703 EVIDENCE FOR DIFFERENTIAL ADAPTATION OF HEPATITIS C VIRUS IN POPULATIONS WITH DIFFERENT HLA-CLASS I-BACKGROUND M. Ruhl1 , T. Knuschke1 , M. Wiese2 , H. Tenckhoff3 , J. Nattermann4 , U. Spengler4 , M. Roggendorf1 , D. Hoffmann5 , J. Timm1 . 1 Institute for Virology, University of Duisburg-Essen, Essen, 2 Gemeinschaftspraxis f¨ ur Verdauungs- und Stoffwechselkrankheiten, 3 University of Leipzig, Leipzig, 4 Department of Medicine I, University of Bonn, Bonn, 5 Centre for Biotechnology, University of Duisburg-Essen, Essen, Germany E-mail: [email protected] Background: Mutational adaptation to HLA-class I-associated selection pressure has been described during Hepatitis C Virus (HCV) infection. However, it is unclear if HCV differentially adapts in populations with different HLA-background. Here, we took advantage of a large HCV genotype 1b outbreak after inoculation of HCV contaminated immunoglobulins in 1977/78 (“East-German Anti-D cohort”) to study this adaptation process and to identify sites under reproducible selection pressure. Methods: Samples from the “East-German Anti-D cohort” are collected since 2007 and HLA-typed. The non-structural proteins were sequenced and mutations were mapped according to their location inside or outside previously described CD8 epitopes to determine the extent of HLA-class I-associated selection pressure. Moreover, residues under reproducible selection pressure in subjects expressing a particular HLA-allele were identified. The frequency of the corresponding polymorphisms in the viral genome was determined in two HLA-diverse populations by analyzing viral sequences from Japan and Central Europe retrieved from a public database. Results: So far, 51 complete sequences covering all nonstructural proteins were obtained. As expected, the frequency of synonymous mutations did not differ between regions inside and outside described CD8 epitopes. However, the frequency of nonsynonymous mutations was significantly higher inside described CD8 epitopes compared to outside (OR 6.2; p < 0.0001). A total of 22 residues have been identified where mutations are reproducibly selected in patients sharing a particular HLA-allele. Interestingly, when the frequencies of the corresponding polymorphisms in isolates from Japan and Central Europe were compared, there was a strong correlation between the difference in HLA-allele-frequency and the difference in the frequency of the polymorphism in these two HLA-diverse populations (r2 = 0.36; p = 0.0034). Conclusion: HLA-class I-associated selection pressure is the major driving force for viral evolution in the non-structural proteins of HCV. Interestingly, we find evidence for differential adaptation of HCV to HLA-class I-mediated immune pressure in two populations with different HLA-background.

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