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667 EARLY DETECTION OF HEPATITIS C USING A ONE MINUTE VISUAL QUALITATIVE BASED KIT SYSTEM IN A COMMUNITY BASED SETTING IN DOHA, QATAR
POSTERS 666 THE EFFECTS OF SCY-635 A NON-IMMUNOSUPPRESSIVE CYCLOSPORIN ANALOG ON STELLATE CELL PROLIFERATION, COLLAGEN SYNTHESIS, TIMP-1 AND COLLAGENASE PRODUCTION B. Scorneaux, G. Thomas, S. Hopkins, R.R. Harris. Biochemistry, Scynexis Incorporated, Durham, NC, USA E-mail: [email protected] Aims: The non-immunosuppressive cyclophilin inhibitor SCY635 is a potent inhibitor of HCV-specific RNA replication in genotype 1 derived HCV replicons. Clinical studies indicate that acute administration of SCY-635 significantly reduces plasma viremia in patients with chronic hepatitis C virus infection. Recent data have shown that cyclosporin and non-immunosuppressive analogues such as NIM-811 can suppress the proliferation of, and collagen production in, hepatic stellate cells (HSC) (Kohjima et al. Liver Int., 2007). To examine the potential effects of SCY-635 on liver fibrosis and apoptosis, we have investigated the effects of SCY-635 on cell growth, collagen synthesis, TIMP-1 and collagenase levels and TGF-b signaling in HSCs. Methods: Human HSC-derived LI-90 cells either resting or stimulated with TGFb1 were used for these studies. The levels of collagen, matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and levels of phosphorylation of p38 and Smad2 were evaluated by ELISA. Cell proliferation (BrdU incorporation) and apoptosis (ApoGlow) were measured. Apoptosis was evaluated using primary rat hepatocytes after treatment with Ad5Ik B adenovirus followed by overnight incubation with TNF-a. Results: SCY-635 and cyclosporin (CsA) decreased the synthesis of collagen. At a clinically relevant concentration of 0.25 mM in nonstimulated or in TGFb1 stimulated LI-90 cells, SCY-635 significantly reduced the collagen production by more than 70%. There was a parallel decrease in TIMP-1 secretion into the HSC medium and an increase in the production of MMP-1. SCY-635 inhibited proliferation without induction of apoptosis. Concentrations of SCY635 ranging from 0.1 to 5 mM enhanced the phosphorylation of p38 and suppressed the phosphorylation of Smad2. SCY-635 inhibition of liver apoptosis was equipotent with CsA exhibiting IC50 values of 2.8±0.3 and 3.6±0.3 mM, respectively. Conclusions: These findings demonstrated that SCY-635 suppressed cell growth, reduced type I collagen synthesis and TIMP-1 secretion, and enhanced MMP-1 production suggesting that SCY-635 may exert an anti-fibrogenic effect. These effects may be mediated through the TGF-b signaling pathway. Taken together, these data suggest that SCY-635 may not only reduce the replication of HCV but also reduce the fibrosis associated with chronic hepatitis C virus infection. 667 EARLY DETECTION OF HEPATITIS C USING A ONE MINUTE VISUAL QUALITATIVE BASED KIT SYSTEM IN A COMMUNITY BASED SETTING IN DOHA, QATAR M. Sharma, K. Matar, N.A. Dweik, S.A. Kaabi, A.K. John, M.A. Mohannadi, M.F. Derbala, A.A. Amin, F. Pasic, R. Yakoob, T. Butt. Hamad Medical Corporation, Doha, Qatar E-mail: [email protected] Background: Early detection and treatment of Hepatitis C can change the natural history of the disease. Aim: The primary aim of this study was to detect infection of hepatitis C using a rapid immunochromatographic assay in a community setting. The secondary aims included assessment of prevalence rate and disease characteristics; including liver function tests, viral load, grade and stage of disease on the liver biopsy. Methods: Two cohorts of 4000 and 3212 people (.004% of the population) were surveyed over a three week period each. Qualitative detection of hepatitis C antibodies was done using a colloidal gold enhanced rapid immunochromatographic assay S260
(Health Chem Diagnostics LLC, FL-USA). Viral load was calculated using RT PCR (Taqman) and those found positive underwent liver biopsy. 28 and 33patients with proven chronic hepatitis C formed the controls for validation of the kit used in the first and the second cohort. Results: 64 people were detected to have positive antibodies (0.008%) to hepatitis C (Cohort 1 – 33/4000, Cohort 2 – 31/3212). In the first cohort, among 28 persons with positive antibodies detected with the kit, abnormal LFT were observed in 21 (75 %) and 10 (35.7 %) had more than >2 ALT levels (Range 17–174 IU/ml). 24 agreed for further testing with RT PCR and another 4 agreed for testing using another method (Enzyme immunoassay). The mean viral load was 5,757,445 IU/ml (Range 27,563–45,448,680 IU/ml). Only one person detected to have positive antibodies with the kit was found to have negative HCV PCR. 15 patients (62.5%) who underwent liver biopsy, 9 showed evidence of ≥F2 fibrosis and 7 moderate necroinflammation (A 2). 12 patients (50%) undertook treatment with Pegylated Interferon and Ribavarin and 8 of them have achieved either rapid or early virological response at the last follow up. The prevalence rate was detected to be 15 per thousand people. The second cohort is under evaluation for further assessment and management. Conclusion: The kit test based detection technique for hepatitis C is quite an effective strategy for early detection especially in community based setting. Further large surveillance studies are required to detect this disease for its effective control. 668 HCV INDUCES ACUTE AND CHRONIC ENDOPLASMIC RETICULUM (ER) STRESS, CAUSING ADAPTATION AND RESISTANCE TO ER STRESS INDUCED APOPTOSIS: A POSSIBLE VIRAL EVASION MECHANISM E. Merquiol1 , S. Mezan1 , M. Mills1 , B. Tirosh2 , O. Shibolet1 . 1 Liver Unit, Division of Medicine, Hadassah-Hebrew University Medical Center, 2 Institute of Drug Research, School of Pharmacology, Faculty of Medicine, Jerusalem, Israel E-mail: [email protected] Background: Hepatitis C virus (HCV) is a positive-strand RNA member of the Flaviviridae family. The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when the ER protein folding capacity is exceeded. This imbalance induces a cyto-protective reaction collectively termed the unfolded protein response (UPR). HCV has been shown to cause acute ER stress. Aim: Characterize the cellular adaptation to HCV-induced chronic ER stress. Methods: The JFH1/HuH7.5 cellular system was used to characterize ER stress induction and UPR pathway activation in response to HCV infection. We used real-time PCR, western blotting, luciferase reporters, immuneflouresence staining, ELISA and FACS. Results: HCV infection induced a wave of acute ER stress starting 2 days and peaking 5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including: IRE1 phosphorylation and XBP-1 splicing. mRNA for the XBP-1 target genes ERdj4 and p58ipk was upregulated by 30±3.7 and 7±0.8 fold respectively. Similarly, eIF2a was phosphorylated and the target genes ATF4, ATF3 and TRB3 were significantly upregulated as compared to baseline. CHOP translocation to the nucleus was observed in HCV infected cells. Only mild activation of ATF6 and BIP was observed using luciferase reporters (2±0.3 and 1.8±0.1 respectively). Interestingly UPR gene mRNA levels did not return to baseline but remained mildly elevated for up to 14 days post infection (2.3±0.8, 5±1.1 and 3.1±0.7 for p58ipk, ATF3 and CHOP respectively). Chronic low level UPR activation was shown to cause adaptation to ER stress. We assessed adaptation of HCV infected cells by stimulation with thapsigargin (Tg). Chronically infected cells showed a marked reduction in XBP-1 splicing in response to
Journal of Hepatology 2010 vol. 52 | S183–S317
(Health Chem Diagnostics LLC, FL-USA). Viral load was calculated using RT PCR (Taqman) and those found positive underwent liver biopsy. 28 and 33patients with proven chronic hepatitis C formed the controls for validation of the kit used in the first and the second cohort. Results: 64 people were detected to have positive antibodies (0.008%) to hepatitis C (Cohort 1 – 33/4000, Cohort 2 – 31/3212). In the first cohort, among 28 persons with positive antibodies detected with the kit, abnormal LFT were observed in 21 (75 %) and 10 (35.7 %) had more than >2 ALT levels (Range 17–174 IU/ml). 24 agreed for further testing with RT PCR and another 4 agreed for testing using another method (Enzyme immunoassay). The mean viral load was 5,757,445 IU/ml (Range 27,563–45,448,680 IU/ml). Only one person detected to have positive antibodies with the kit was found to have negative HCV PCR. 15 patients (62.5%) who underwent liver biopsy, 9 showed evidence of ≥F2 fibrosis and 7 moderate necroinflammation (A 2). 12 patients (50%) undertook treatment with Pegylated Interferon and Ribavarin and 8 of them have achieved either rapid or early virological response at the last follow up. The prevalence rate was detected to be 15 per thousand people. The second cohort is under evaluation for further assessment and management. Conclusion: The kit test based detection technique for hepatitis C is quite an effective strategy for early detection especially in community based setting. Further large surveillance studies are required to detect this disease for its effective control. 668 HCV INDUCES ACUTE AND CHRONIC ENDOPLASMIC RETICULUM (ER) STRESS, CAUSING ADAPTATION AND RESISTANCE TO ER STRESS INDUCED APOPTOSIS: A POSSIBLE VIRAL EVASION MECHANISM E. Merquiol1 , S. Mezan1 , M. Mills1 , B. Tirosh2 , O. Shibolet1 . 1 Liver Unit, Division of Medicine, Hadassah-Hebrew University Medical Center, 2 Institute of Drug Research, School of Pharmacology, Faculty of Medicine, Jerusalem, Israel E-mail: [email protected] Background: Hepatitis C virus (HCV) is a positive-strand RNA member of the Flaviviridae family. The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when the ER protein folding capacity is exceeded. This imbalance induces a cyto-protective reaction collectively termed the unfolded protein response (UPR). HCV has been shown to cause acute ER stress. Aim: Characterize the cellular adaptation to HCV-induced chronic ER stress. Methods: The JFH1/HuH7.5 cellular system was used to characterize ER stress induction and UPR pathway activation in response to HCV infection. We used real-time PCR, western blotting, luciferase reporters, immuneflouresence staining, ELISA and FACS. Results: HCV infection induced a wave of acute ER stress starting 2 days and peaking 5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including: IRE1 phosphorylation and XBP-1 splicing. mRNA for the XBP-1 target genes ERdj4 and p58ipk was upregulated by 30±3.7 and 7±0.8 fold respectively. Similarly, eIF2a was phosphorylated and the target genes ATF4, ATF3 and TRB3 were significantly upregulated as compared to baseline. CHOP translocation to the nucleus was observed in HCV infected cells. Only mild activation of ATF6 and BIP was observed using luciferase reporters (2±0.3 and 1.8±0.1 respectively). Interestingly UPR gene mRNA levels did not return to baseline but remained mildly elevated for up to 14 days post infection (2.3±0.8, 5±1.1 and 3.1±0.7 for p58ipk, ATF3 and CHOP respectively). Chronic low level UPR activation was shown to cause adaptation to ER stress. We assessed adaptation of HCV infected cells by stimulation with thapsigargin (Tg). Chronically infected cells showed a marked reduction in XBP-1 splicing in response to
Journal of Hepatology 2010 vol. 52 | S183–S317