700 INFLAMMASOME IS MODULATED BY PEGYLATED INTERFERON MONOTHERAPY IN CHRONIC HEPATITIS C PATIENTS

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

Journal of Hepatology 2010 vol. 52 | S183–S317