IL-17A is considered to guide liver inflammation and fibrosis. From twenty-two human liver samples of different fibrosis stages (F0 to F4), IL-17A, IL-22, and TGFβ1 protein expression in liver tissue lysates were analyzed. Ten paired samples of liver tissue (F0–F1 stage) and blood from the same patient were used to analyze intrahepatic and blood T-lymphoid IL-17A+ cells by flow cytometry. The analyses have been performed regardless of pathology, considering the stage of fibrosis. Human liver tissue was used for the primary human liver slice cultures, followed by subsequent cytokine stimulation and fibrotic markers’ analysis by ELISA. IL-17A production in human liver tissue was significantly higher in the early fibrotic stage compared with the advanced stage. Th17 T cells and, to a lesser extent, MAIT cells were the main sources of IL-17A in both compartments, the liver and the blood. Moreover, the presence of liver Th17IL-17A+INFγ+ cells was detected in the liver. IL-17A stimulation of human liver slice culture increased the expression of profibrotic and pro-inflammatory markers. IL-17A, secreted by Th17 and MAIT cells in the liver, triggered fibrosis by inducing the expression of IL-6 and profibrotic markers and could be a target for antifibrotic treatment. Further amplitude studies are needed to confirm the current results.

BACKGROUND:Liver fibrosis can result in end-stage liver failure and death.AIM:To examine human liver fibrogenesis and anti-fibrotic therapies, we evaluated the three dimensional ex vivo liver slice (LS) model.METHODS:Fibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected from patients after liver resection. Human liver slices (HLS) were cultivated for up to 21 days. Hepatitis C virus (HCV) infection, alcohol (ethanol stimulation) and steatosis (palmitate stimulation) were examined in fibrotic (F2 to F4) liver slices infected (or not) with HCV. F0-F1 HLS were used as controls. At day 0, either ursodeoxycholic acid (choleretic and hepatoprotective properties) and/or α-tocopherol (antioxidant properties) were added to standard of care on HLS and fibrotic liver slices, infected (or not) with HCV. Expression of the biomarkers of fibrosis and the triglyceride production were checked by quantitative reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay.RESULTS:The cultures were viable in vitro for 21 days allowing to study fibrosis inducers and to estimate the effect of anti-fibrotic drugs. Expression of the biomarkers of fibrosis and the progression to steatosis (estimated by triglycerides production) was increased with the addition of HCV and /or ethanol or palmitate. From day 15 of the follow-up studies, a significant decrease of both transforming growth factor β-1 and Procol1A1 expression and triglycerides production was observed when a combined anti-fibrotic treatment was applied on HCV infected F2-F4 LS cultures.CONCLUSION:These results show that the human three dimensional ex vivo model effectively reflects the in vivo processes in damaged human liver (viral, alcoholic, nonalcoholic steatohepatitis liver diseases) and provides the proof of concept that the LS examined model permits a rapid evaluation of new anti-fibrotic therapies when used alone or in combination.

Background: To study the first steps of liver molecular fibrogenesis in our ex vivo model of human liver slices culture. Methods: Human fibrotic liver samples (F0 to F4 fibrosis stage according to the METAVIR score) were collected after liver resection. Human liver slices of 350 µm (2.7x106cells per slice) were cultivated for up to 21 days. Fibrotic liver slices F2 to F4 (FLS) were infected (or not) with HCVcc supernatant infection [Con1/C3 (genotype1b)] (MOI = 0.1) (INF FLS). F0-F1 HLS (HLS) were used as controls. At day 0, either ursodeoxycholic acid (UDCA, only choleretic and hepatoprotective properties) and/or atocopherol (Toco, anti-oxydant properties which could reduce fibrosis progression) were added to standard of care concentrations on HLS and FLS. The following fibrosis markers expression were assayed in HLS, in FLS and in INF FLS, [tumor growth factor b (TGFβ), Heat shock protein 47 (Hsp47), Alpha smooth muscle actin (α-Sma), Procollagen1 A1 (Procl1A1), Matrix metalloproteinases 2, 9 (MMP-2, 9), Vascular Endothelial Growth Factor (VEGF)] and checked by RT-qPCR and the triglyceride production by ELISA assays. Results: The TGFβ, Hsp47,α-Sma, Procl1A1, MMP-2, MMP-9, VEGF expression and the triglyceride production increased significantly in both HLS and FLS but to a higher extent in FLS as compared to HLS, with a triglyceride production higher [~x 3.2] in FLS. HCV infection increased significantly the fibrotic markers expression and triglyceride production in non-infected (NI) FLS and INF FLS to a higher extent in INF FLS as compared to controls. The induced molecular fibrogenesis significantly increased, particularly true for the advanced stage of fibrosis, including cirrhosis. Under a-tocopherol exposure, a significant reduction of TGF-β RNA expression was observed in NINF and INF FLS but only for the stage F4. UDCA did not impact TGFβ RNA expression in NINF F0 to F4 liver slices, but significantly reduces its expression in F2F3F4 infected liver slices. Combination of UDCA and Toco treatments did not result in an additive effect and only reduced the TGFβ RNA expression in F2-F4 INF HLS but not in F2-F4 NI HLS. Conclusion: Our ex vivo model of human liver slices culture, supporting hepatocyte-specific gene expression for 21 days, provides an easy tool for studying human molecular liver fibrogenesis which could accelerate the evaluation of the potency of new anti-fibrotic therapies, alone or in combination.

AASLD Publications Hepatology Liver Transplantation Clinical Liver Disease Hepatology Communications AASLD Publications Hepatology Liver Transplantation Clinical Liver Disease Hepatology Communications

AIM:To evaluate the antiviral potency of a new anti-hepatitis C virus (HCV) antiviral agent targeting the cellular autophagy machinery. METHODS:Non-infected liver slices, obtained from human liver resection and cut in 350 μm-thick slices (2.7 × 10(6) cells per slice) were infected with cell culture-grown HCV Con1b/C3 supernatant (multiplicity of infection = 0.1) cultivated for up to ten days. HCV infected slices were treated at day 4 post-infection with GNS-396 for 6 d at different concentrations. HCV replication was evaluated by strand-specific real-time quantitative reverse transcription - polymerase chain reaction. The infectivity titers of supernatants were evaluated by foci formation upon inoculation into naive Huh-7.5.1 cells. The cytotoxic effect of the drugs was evaluated by lactate dehydrogenase leakage assays. RESULTS:The antiviral efficacy of a new antiviral drug, GNS-396, an autophagy inhibitor, on HCV infection of adult human liver slices was evidenced in a dose-dependent manner. At day 6 post-treatment, GNS-396 EC50 was 158 nmol/L without cytotoxic effect (compared to hydroxychloroquine EC50 = 1.17 μmol/L). CONCLUSION:Our results demonstrated that our ex vivo model is efficient for evaluation the potency of autophagy inhibitors, in particular a new quinoline derivative GNS-396 as antiviral could inhibit HCV infection in a dose-dependent manner without cytotoxic effect.

BACKGROUND AND AIMS: Interferon-gamma (IFN-c) is a dimerized soluble cytokine which is critical to the defense against HCV infection as it can inhibit HCV replication in the replicon system.It has been proven that polymorphisms in some genes may influence the persistence of HCV infection, clinical outcome, HCV replication, and liver damage.METHODS: Family-based association designs offer a compromise between traditional linkage studies and case-control association studies.In these studies, association is assessed within families, and hence the confounding due to population heterogeneity can be eliminated.In our study 177 families have been recruited from The Upper, Middle, and Lower Egypt governments.These families include 323HCV patients as well as 347 non infected individuals.All were subjected to Routine clinical & laboratory investigations in addition to PCR HCV, HCV genotyping.Polymorphism of IFN-c (+874T/A, rs 62559044) was genotyped using ARM-PCR and confirmed using direct sequencing specific primers.RESULTS: Groups of our study were in Hardy-Weinberg equilibrium for IFN-c (+874T/A, rs 62559044).The T of IFN-c (+874T) allele was associated with 2 folds increased risk for HCV patients as compared to non-infected exposed person (OR = 1.9597, 95% CI 1.5269-2.5152,z statistic 5.284, p < 0.0001).The distribution of INF-c polymorphism was 10.81% AA, 51.39% AT &37.77%TT in HCV infected patients while in HCV-negative patients the distribution was 35.73% AA, 52.17% AT& 12.1%TT CONCLUSIONS: The T allele of rs 62559044 SNP of +IFNc gene polymorphism may have a crucial role of susceptibility to HCV infection in Egyptian families.

BackgroundChronic hepatitis C is a major cause of liver fibrosis and cirrhosis. It is generally accepted that inflammation that occurs in response to hepatocyte infection by the hepatitis C virus (HCV) is the main mechanism that triggers myofibroblast differentiation and stimulation in chronic hepatitis C. The aim of this study was to determine if HCV might infect human liver myofibroblasts (HLMF) and directly stimulate their fibrogenic activities.MethodsWe evaluated the expression of the viral entry receptors, levels of HCV-RNA and HCV-protein and the expression of fibrosis markers in HLMF by using quantitative PCR, western blot and immunofluorescence analyses. Pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) were used to study the ability of HLMF to support viral entry, replication and fibrosis induction.ResultsWe showed that HLMF expressed all known molecules of the HCV receptor complex, i.e. CD81, LDL-R, scavenger receptor-BI, claudin-1 and occludin. These cells were also permissive to HCVpp entry. Inoculation with HCVcc caused short-term infection of these cells, as shown by their content in positive-and negative-strand HCV RNA, in core and NS3 viral proteins, and by their release of core protein levels in the culture supernatants. HCV infection stimulated myofibroblastic differentiation, proliferation and collagen production in these cells. In addition, evidence of in vivo infection was provided by the detection of positive- and negative-strand HCV RNA in preparations of HLMF obtained from HCV-infected patients.ConclusionThese findings indicate that HCV infection of HLMF can occur and trigger extracellular matrix overproduction, thereby contributing to the development of HCV-related liver fibrosis.

expression in liver biopsies and serum specimens obtained sequentially from the acute phase of HCV infection in chimpanzees to determine the kinetics of miR-122 and their relationship to HCV replication, interferon (IFN) type I response and liver pathology.Material and methods: Liver biopsies and sera were obtained from three chimpanzees infected with genotype 1a HCV and observed over a period up to 201 days after inoculation (DAI).Chimpanzees started to have HCV RNA detected in serum from DAI 4 to 7. Two chimpanzees (CH6413 and CH256) remained HCV RNA-positive throughout the study period, one (CH1541) cleared the infection by DAI 131.Hepatic and serum levels of miR-122 and hepatic levels of IFN-a, IFN-b and 2 5 oligoadenylate synthetase-2 (2,5-OAS-2) mRNAs were measured by real-time PCR.Results: Hepatic levels of miR-122 decreased to approximately 50% of preinoculation values at the peak of HCV replication in series of 5 to 12 consecutive weekly biopsies and returned to initial levels during the period when HCV RNA titers were declining (DAI 75 to 201 in CH256, DAI 50 to 201 in CH6413, DAI 61 to 131 in CH1541).This inverse correlation was statistically significant in two chimpanzees (r = -0.681;p = 0.015 in CH256; r = -0.098,p = 0.003 in CH1541).In all 3 chimpanzees, the decrease in hepatic miR-122 was associated with rise in levels of hepatic IFN-b and INF-a and 2,5-OAS-2 mRNA.The profile of miR-122 in serum correlated positively with serum ALT activity values (r = 0.75 to 0.9; p ≤ 0.002).Conclusions: In the chimpanzee model, negative correlations between miR-122 expression in the liver and HCV replication together with evidence of IFN type I induction contribute to molecular pathogenesis of the acute phase on HCV infection.Changes of miR-122 in serum positively correlated with profile of ALT activity and suggested that serum miR-122 may be a biomarker of HCV-associated liver injury.

The development of human cultured hepatitis C virus (HCV) replication‐permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however, this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high‐titer infectious virus as well as Japanese fulminant hepatitis type 1, H77/C3, and Con1/C3. This experimental model was employed to demonstrate HCV neutralization or HCV inhibition, in a dose‐dependent manner, either by cluster of differentiation 81 or envelope protein 2–specific antibodies or convalescent serum from a recovered HCV patient or by antiviral drugs. Conclusion: This new ex vivo model represents a powerful tool for studying the viral life cycle and dynamics of virus spread in native tissue and also allows one to evaluate the efficacy of new antiviral drugs. (HEPATOLOGY 2012;56:861–872)

Background and Aims: PD-1 is a member of the extended CD28/CTLA-4 family of T cell regulators and negatively regulates immune responses.Interferon-regulated gene expression is necessary to mount an effective antiviral response for HCV clearance.We investigated the proliferation, activation and functional status of CD8 + T lymphocytes along with status of PD-1 expression and role of IRGs in CHC.Methods: Patients with histological evidence of CHC(n = 15), spontaneously resolved HCV patients (SR-C, n = 5) and healthy controls (HC, n = 5) were included.Proliferative capacity, cytotoxicity and IFNg production by HCV-specific CD8 + T cells were tested after stimulation with HCV peptide pools.PD-1 expression analysed by flow cytometry.Total RNA was isolated from PBMCs and gene expression analysis of PKR, OAS1, IP-10, USP-18, IRF1, IRF7 and IFI27 was performed by RT-PCR.Results: The frequency of CD8+T cells proliferating after HCV peptide stimulation was higher in CHC than HC (0.314±0.27 vs. 0.088±0.03,P < 0.05)., but the frequency of CD8 + T/ IFNg + cells was 3 fold lower in CH-C patient.The spontaneously cleared subjects displayed a significantly higher frequency of CD8 + T/ IFNg + cells (8.653±0.32%).The fraction of CD8 + T cells showing cytotoxic capacity was lower in CH-C patients as compared to SR-C (0.74±0.531 vs. 2.37±1.4,P < 0.05).PD-1 expression was found to be higher on CH-C patients as compared to HC (0.82±0.61 vs 0.005±0.005,P < 0.05).IP-10 and IRF-1 expression were higher in CH-C patients as compared to SR-C, suggestive of an ineffective and rather injurious attempt by the host immune system of CH-C patients. Conclusions:The proliferative capacity of CD8 + T cells was increased but IFN-g production was lower in CH-C patients as compared to HC. II) A significant decrease in cytotoxic capacity of CD8 + T cells was observed in CH-C in contrast to SR-C, indicating a robust immune response in subjects who had spontaneously cleared the virus.Higher expression of PD-1 was associated with HCV persistence, demonstrating the dysfunctionality of CD8 + T cells.IV) The expressions of IP-10 and IRF-1 suggest that along with dysfunctional CD8 + T lymphocytes, the increased gene expressions of important IRGs were unable to control the viral infection leading to chronicity.

05G. VIRAL HEPATITIS -G) HEPATITIS C -CLINICAL (THERAPY) S307recombinant protein comprising IFNalfa-2b genetically fused to human albumin, in combination with oral RBV in IFN treatment-naive, genotype 1, CHC patients.HRQOL was assessed by changes in SF-36 v2 and missed workdays due to CHC in patients treated with alb-IFN vs PEG-IFNalfa-2a.Methods: 458 patients were randomized to 4 subcutaneous treatment groups: PEG-IFN 180mcg qwk, and alb-IFN 900 and 1200mcg q2wk, and 1200mcg q4wk.Primary efficacy endpoint: SVR.SF-36 was evaluated at baseline, wk4, 12, 24, and 48 on treatment, and wk4, 12, and 24 posttreatment.Patient disability was evaluated by number of missed workdays or days with impaired activity.Missing data were handled using the last observation carried forward.Results: By ITT analysis, SVR rates were 58.5% for alb-IFN 900mcg, 55.5% and 50.9% for 1200mcg q2wk and q4wk, respectively, and 57.9% for PEG-IFN (P = NS).Overall, alb-IFN had less impact on the physical and mental domains of SF 36 during treatment vs PEG-IFN.Reductions in SF-36 scores occurred by wk4-12, consistent with the occurrence of most treatment-associated AEs.At wk12, there was significantly less worsening in SF-36 physical (P = 0.04) and mental (P = 0.02) component scores, as well as in role-physical (P = 0.02), bodily pain (P = 0.003), vitality (P = 0.02), social-functioning (P < 0.001), and mental-health (P = 0.003) domain scores with alb-IFN 900mcg vs PEG-IFN.Overall, disability day assessments were significantly more favorable with alb-IFN 900mcg vs PEG-IFN.The proportion of patients with 7 missed workdays in the month prior to assessment was significantly lower with alb-IFN 900mcg vs PEG-IFN at wk12 (4.2% vs 18.1%; P = 0.006) and 24 (5.3% vs 20.3%: P = 0.005).The results for alb-IFN 1200mcg q2wk and q4wk were generally comparable to those for PEG IFN.Conclusions: The alb-IFN 900mcg q2wk regimen was associated with significantly more favorable HRQOL and fewer missed workdays, while maintaining efficacy at least comparable to that of PEG-IFN.