Liver Hepatitis C Virus Infection Research Articles

Serum microRNA-16 as a potential biomarker for HCV-induced hepato-cellular carcinoma in Egyptian patients

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world. Two risk factors that cause 80–90% of HCC cases globally are chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV). The diagnostic value of circulating microRNAs (miRNAs) in numerous tumors has been described. Our research assessed microRNA-16 (miR-16) as a novel biomarker in patients with HCV-induced HCC. The study included three groups. Group 1 included 55 individuals with cirrhosis caused by liver HCV infection in addition to HCC. Group 2 included 55 individuals with cirrhosis brought on by HCV infection. Group 3 included 55 normal control individuals. Expression of miR-16 in blood was assessed by real-time polymerase chain reaction (RT-PCR). The mean level of miR-16 was significantly different in the three groups, with group 1 having the greatest value (1.098 ± 0.647), followed by group 2 (1.1035 ± 0.8567) and group 3 (control subjects) having the lowest value (0.3842 ± 0.21485). The receiver operating characteristic (ROC) curve analysis showed that miR-16 had a higher diagnostic value at area under the curve (AUC) of 0.935 than alpha-feto protein (AUC of 0.859) to differentiate between HCC and control subjects. MiR-16 has a sensitivity of 81.82 % and a specificity of 69.09%, to distinguish between patients with liver cirrhosis and HCC patients. Our findings illustrated that circulating miR-16 can be proposed as a marker for detection of patients with HCV-induced HCC.

Codon usage of human hepatitis C virus clearance genes in relation to its expression.

Hepatitis C virus (HCV) infection is among the leading causes of hepatocellular carcinoma and liver cirrhosis globally, with a high economic burden. The disease progression is well established, but less is known about the spontaneous HCV infection clearance. This study tries to establish the relationship between codon biasness and expression of HCV clearance candidate genes in normal and HCV infected liver tissues. A total of 112 coding sequences comprising 151 679 codons were subjected to the computation of codon indices, namely relative synonymous codon usage, an effective number of codon (Nc), frequency of optimal codon, codon adaptation index, codon bias index, and base compositions. Codon indices report of GC3s, GC12, hydropathicity, and aromaticity implicates both mutational and translational selection in the candidate gene set. This was further correlated with the differentially expressed genes among the selected genes using BioGPS. A significant correlation is observed between the gene expression of normal liver and cancerous liver tissues with codon bias (Nc). Gene expression is also correlated with relative codon bias values, indicating that CCL5, APOA2, CD28, IFITM1, and TNFSF4 genes have higher expression. These results are quite encouraging in selecting the high responsive genes in HCV clearance. However, there could be additional genes which could also orchestrate the clearance role with the above mentioned first line of defensive genes.

Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals.

The increasing worldwide prevalence of Hepatocellular carcinoma (HCC), characterized by resistance to conventional chemotherapy, poor prognosis and eventually mortality, place it as a prime target for new modes of prevention and treatment. Hepatitis C Virus (HCV) is the predominant risk factor for HCC in the US and Europe. Multiple epidemiological studies showed that sustained virological responses (SVR) following treatment with the powerful direct acting antivirals (DAAs), which have replaced interferon-based regimes, do not eliminate tumor development. We aimed to identify an HCV-specific pathogenic mechanism that persists post SVR following DAAs treatment. We demonstrate that HCV infection induces genome-wide epigenetic changes by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) for histone post-translational modifications that are epigenetic markers for active and repressed chromatin. The changes in histone modifications correlate with reprogramed host gene expression and alter signaling pathways known to be associated with HCV life cycle and HCC. These epigenetic alterations require the presence of HCV RNA or/and expression of the viral proteins in the cells. Importantly, the epigenetic changes induced following infection persist as an "epigenetic signature" after virus eradication by DAAs treatment, as detected using in vitro HCV infection models. These observations led to the identification of an 8 gene signature that is associated with HCC development and demonstrate persistent epigenetic alterations in HCV infected and post SVR liver biopsy samples. The epigenetic signature was reverted in vitro by drugs that inhibit epigenetic modifying enzyme and by the EGFR inhibitor, Erlotinib. This epigenetic “scarring” of the genome, persisting following HCV eradication, suggest a novel mechanism for the persistent pathogenesis of HCV after its eradication by DAAs. Our study offers new avenues for prevention of the persistent oncogenic effects of chronic hepatitis infections using specific drugs to revert the epigenetic changes to the genome.

P53 and STAT3 Dependent Expression of a Novel microRNA Restricts Hepatitis C Virus Infection Through Hyper-β-Oxidation of Fatty Acids by Impeding Cytosolic CAP350

Background: The underlying complex molecular mechanism of Hepatitis C virus (HCV) mediated liver disease progression is still unresolved. Next generation sequencing of small RNAs provides genome-wide data about regulatory microRNAs. Here, we have identified undiscovered low abundant regulatory miRNAs and investigated the role of a novel human liver specific miRNAs in HCV mediated liver disease development. Methods: To discover unannotated miRNAs, unmapped sequences from small RNA transcriptome profiling of HCV infected and un-infected liver tissues were subjected to miRDeep2.pl pipeline. Targets predicted using MiRanda and Ingenuity pathway analysis (IPA). Northern-blot, Dicer knock down (K/D), Ago2-RNA immunoprecipitation (RIP), Stem-loop Real-time PCR (SLRTPCR), immunoblot (IB) analysis, luciferase assay, Chromatin immunoprecipitation (ChIP) etc. were used as required. Findings: Among seven predicted novel miRNAs, miR-c12 was found human liver specific miRNA. Dicer K/D and RIP assays showed that miR-c12 followed canonical miRNA biogenesis pathways. Its low expression in chronic HCV infected (CHC) tissues than normal liver indicated its protective role against HCV infection. Furthermore, upon HCV infection downregulation of p53 expression and hypermethylation of STAT3 binding site in the promoter of miR-c12 restricted its expression in CHC. IPA analysis revealed miR-c12 controlled mostly the fatty acid metabolism pathway. Reduced triglyceride accumulation and enhanced PPARα mediated fatty-acid catabolism upon miR-c12 retention in cells by targeting CAP350, a suppressor of PPARα, confirmed its role in lipid metabolism pathways. Interpretations: We discovered a previously unidentified low abundant human-liver specific miRNA having role in lipid homeostasis maintenance. Hence, miR-c12 together with anti-virals might be more effective therapeutic in restricting HCV infection. Funding Statement: Department of Biotechnology (DBT), Government of India [BT/PR11971/MED/29/890/2014]. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Ethics committee of the Institute of Post Graduate Medical Education and Research (IPGME&R), Kolkata, India and Indraprastha Apollo Hospital, New Delhi had approved the study. Blood and tissues were collected from patients who were willing to participate in the study with prior written consent.

Complement Regulation and Immune Evasion by Hepatitis C Virus.

A prominent role for complement has been identified in the linkage of innate and adaptive immunity. The liver is the main source of complement and hepatocytes are the primary sites for synthesis of complement components in vivo. We have discovered that hepatitis C virus (HCV) impairs C4 and C3 synthesis. Liver damage may diminish capacity of complement synthesis in patients. However, we observed that the changes in measured complement components in chronically HCV infected patients do not correlate with liver fibrosis or rheumatoid factor present in the blood, serum albumin, or alkaline phosphatase levels. Complement component C3 is of critical importance in B cell activation and T cell-dependent antibody responses. C3 activity is required for optimal expansion of CD8+T cells during a systemic viral infection. Deficiencies in complement may predispose patients to infections via ineffective opsonization, and defects in lytic activity via membrane attack complex. Interestingly, C9 is significantly reduced at the mRNA level in chronically HCV infected liver biopsy specimens, while many hepatocyte derived complement components (C6, C8, Factor B, MASP1, and MBL) and unrelated genes remain mostly unaffected. This implies an HCV specific effect, not a global effect from liver disease.

Interaction of miR-125b-5p with Human antigen R mRNA: Mechanism of controlling HCV replication

Cellular miRNAs influence Hepatitis C virus (HCV) infection in multiple ways. In this study, we demonstrate that miR-125b-5p is upregulated in HCV infected patient serum samples as well as in HCV infected liver carcinoma cells and is involved in translational regulation of one of its predicted targets, Human antigen R (HuR). We used miRNA mimics and antagomiRs to confirm that HuR is a bonafide miR-125b target. Previously, we have shown that HuR is a positive regulator of HCV replication, whereas we noticed that miR-125b is a negative regulator of HCV infection. As a connecting link between these two observations, we showed that knockdown of miR-125b-5p increased HuR protein levels and rescued HCV replication when the availability of HuR in the cytoplasm was compromised using siRNAs against HuR or an inhibitor of HuR export to the cytoplasm. Overall, the study sheds light on the ability of host cell to use a miRNA as a tool to control virus propagation.

Determining major genotypes of hepatitis C virus among transplant recipients by real-time polymerase chain reaction assay.

Background:Hepatitis C virus (HCV) infection still exists as a health concern among the transplant patients. Because of the severity of the disease, different responses to treatment, and side effects resulting from long therapeutic period, determination of genotypes and viral loads can help choose the best treatment protocols.Objectives:This study aimed to determine the HCV genotypes and its distribution patterns among liver, kidney, and bone marrow recipient candidates across Iran, referred to Namazi Hospital, southern Iran.Patients and Methods:A total of 101 individuals, including 44 (43.6%) liver, 55 (54.5%) kidney, and 2 (2%) bone marrow recipient candidates, with ages ranging between 5 and 74 years (Mean ±SD: 46.53 ± 13.73 y) participated in this study. From those, whole blood sample were collected and anti-HCV antibodies, RNA detection, and genotyping were performed on plasma using commercial chromatographic immunoassay, TaqMan one-step real-time polymerase chain reaction (RT-PCR), and genotyping RT-PCR kits, respectively. The frequencies of anti-HCV antibodies, RNA, various genotypes, and the viral load were compared with respect to gender, age, and transplant recipient groups.Results:Of 101 individuals, 47 (46.5%) were positive for anti-HCV antibodies and 34 (33.7%) for RNA with a significant difference (P < 0.05). RNA copy number ranged from 4.6 × 103 to 3.11 × 107 copies/mL, median: 2.92 × 106 copies/mL, with no statistical differences in all groups. Analyses revealed no significant differences between the frequencies of anti-HCV antibodies or RNA in different groups. The frequencies of the genotypes 1 (50%) and 3 (35.3%) were higher than those of the genotypes 2 (2.9%), 4 (2.9%), and undetermined one (8.8%). Genotype 1 was significantly more prevalent in liver transplant recipients, those older than 40 years, and male cases (P < 0.05).Conclusions:Considering the high frequency of genotypes 1 and 3 among the studied groups, it is suggested that before and after transplantation programs be improved to manage and treat the disease efficiently, based on the standard protocols for such genotypes in the region. Accordingly, the occurrence of post-transplant complications due to immunosuppression among all the recipients as well as reinfection in HCV infected liver transplant patients can be diminished.

Impaired expression of type I and type II interferon receptors in HCV-associated chronic liver disease and liver cirrhosis.

PurposeChronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients.MethodsPrimary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting.ResultHCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rβ (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients.ConclusionHCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.

Influence of Calcineurin Inhibitors Selection On the Outcome of HCV Recurrence in Liver Transplantation Recipients: A Meta-Analysis.

Background and Objective A controversy exists over whether the selection of calcineurin inhibitors (CNIs), namely Tacrolimus (Tac) or Cyclosporine A (CsA), can influence the outcomes of liver transplant recipients with hepatitis C virus (HCV) infection, especially the outcome of HCV recurrence. Here, we performed a meta-analysis on the outcomes of HCV infected liver transplant recipients treated with Tac or CsA. Methods We performed an electronic database search as well as a manual search of relevant journals until the end of 2012. The selection criteria were randomized controlled trials (RCT) with Tac and CsA treatment arms in which HCV-infected liver transplant recipients were included in the study population. Two authors identified and extracted data independently. Dichotomous data were expressed as relative risks (RR) and 95% confidence intervals (CI). Results In all, eight RCTs with 734 patients were included in this study. No significant heterogeneity was detected among the pooled data (all P values > 0.1) and a fixed effects model was used to calculate the overall treatment effect size. Statistically significant differences between Tac and CsA groups were not found for the incidences of severe fibrosis (RR = 0.98, 95% CI = 0.59-1.62, P = 0.93), fibrosing cholestatic hepatitis (RR = 0.98, 95% CI 0.49-1.95, P = 0.95), patient mortality (RR = 0.70, 95% CI 0.49-1.01, P = 0.06) and graft survival (RR = 0.88, 95% CI 0.64-1.23, P = 0.46). Regarding the aspect of rejection, Tac group exhibited a lower incidence of acute rejection than CsA group (RR = 0.68, 95% CI 0.49-0.95, P = 0.02).Figure: No Caption available.Figure: No Caption available.Conclusion: CNIs selection did not influence the outcome of liver transplant recipients with HCV infection. However, Tac can offer advantages over CsA in lowering the incidence of acute rejection.

Promoter hypermethylation of Wnt pathway inhibitors in hepatitis C virus - induced multistep hepatocarcinogenesis.

BackgroundAberrant DNA methylation profiles are a characteristic feature of almost all types of cancers including hepatocellular carcinoma (HCC) and play an important role in carcinogenesis. In spite of the accumulating evidence that suggests appearance of such aberrations at precancerous stages, very little effort has been invested to investigate such possible methylation events in patients at risk of developing HCC i.e. those suffering from chronic hepatitis C virus (HCV) infection and liver cirrhosis (LC). We reasoned that such an analysis could lead to the identification of novel predictive biomarkers as well as potential drug targets.MethodsPromoter methylation status of two Wnt inhibitors SFRP2 and DKK1 was quantitatively analyzed by bisulfite pyrosequencing in a series of liver biopsy samples. These biopsies were collected from HCV-infected individuals suffering from chronic hepatitis (CH; n = 15), liver cirrhosis (LC; n = 13) and hepatocellular carcinoma (HCC; n = 41). DNA isolated from infection free normal livers (N; n =10) was used as control.ResultsOur analysis revealed that both of the genomic loci were significantly hypermethylated in CH patients’ livers as compared to normal controls (p = 0.0136 & 0.0084 for SFRP2 and DKK1, respectively; Mann–Whitney U test). DNA methylation levels for both loci were also significantly higher in all the diseased cohorts as compared to normal controls (p < 0.0001 and = 0.0011 for SFRP2 and DKK1, respectively; Kruskal-Wallis test). However, a comparison between three disease cohorts (CH, LC & HCC) revealed no significant difference in levels of DNA methylation at DKK1 promoter. In contrast, a progressive increase in DNA methylation levels was observed at the SFRP2 promoter (i.e. N < CH & LC < HCC).ConclusionThis study demonstrated that in HCV infected liver tissues hypermethylation at promoter regions of key cancer related genes SFRP2 and DKK1, appears early at CH and LC stages, long before the appearance of HCC.

Hepatitis C Virus Induced miR200c Down Modulates FAP-1, a Negative Regulator of Src Signaling and Promotes Hepatic Fibrosis

Hepatitis C virus (HCV) induced liver disease is the leading indication for liver transplantation (LTx). Reinfection and accelerated development of fibrosis is a universal phenomenon following LTx. The molecular events that lead to fibrosis following HCV infection still remains poorly defined. In this study, we determined microRNA (miRNA) and mRNA expression profiles in livers from chronic HCV patients and normals using microarrays. Using Genego software and pathway finder we performed an interactive analysis to identify target genes that are modulated by miRNAs. 22 miRNAs were up regulated (>2 fold) and 35 miRNAs were down regulated (>2fold) compared to controls. Liver from HCV patients demonstrated increased expression of 306 genes (>3 fold) and reduced expression of 133 genes (>3 fold). Combinatorial analysis of the networks modulated by the miRNAs identified regulation of the phospholipase C pathway (miR200c, miR20b, and miR31through cellular proto-oncogene tyrosine-protein kinase Src (cSrc)), response to growth factors and hormones (miR141, miR107 and miR200c through peroxisome proliferator-activated receptor alpha and extracellular-signal-regulated kinases, and regulation of cellular proliferation (miR20b, miR10b, and miR141 through cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1 p21). Real time PCR (RT-PCR) validation of the miRNA in HCV infected livers demonstrated a 3.3 ±0.9 fold increase in miR200c. In vitro transfection of fibroblasts with miR200c resulted in a 2.2 fold reduction in expression of tyrosine-protein phosphatase non-receptor type 13 or FAS associated phosphatase 1 (FAP-1) and 2.3 fold increase in expression of cSrc. miR200c transfection resulted in significant increases in expression of collagen and fibroblast growth factor (2.8 and 3.4 fold, p<0.05). Therefore, we propose that HCV induced increased expression of miR200c can down modulate the expression of FAP1, a critical regulator of Src and MAP kinase pathway that play an important role in the production of fibrogenic growth factors and development of fibrosis.

ENCODE Tiling Array Analysis Identifies Differentially Expressed Annotated and Novel 5′ Capped RNAs in Hepatitis C Infected Liver

Microarray studies of chronic hepatitis C infection have provided valuable information regarding the host response to viral infection. However, recent studies of the human transcriptome indicate pervasive transcription in previously unannotated regions of the genome and that many RNA transcripts have short or lack 3′ poly(A) ends. We hypothesized that using ENCODE tiling arrays (1% of the genome) in combination with affinity purifying Pol II RNAs by their unique 5′ m7GpppN cap would identify previously undescribed annotated and unannotated genes that are differentially expressed in liver during hepatitis C virus (HCV) infection. Both 5′-capped and poly(A)+ populations of RNA were analyzed using ENCODE tiling arrays. Sixty-four annotated genes were significantly increased in HCV cirrhotic as compared to control liver; twenty-seven (42%) of these genes were identified only by analyzing 5′ capped RNA. Thirty-one annotated genes were significantly decreased; sixteen (50%) of these were identified only by analyzing 5′ capped RNA. Bioinformatic analysis showed that capped RNA produced more consistent results, provided a more extensive expression profile of intronic regions and identified upregulated Pol II transcriptionally active regions in unannotated areas of the genome in HCV cirrhotic liver. Two of these regions were verified by PCR and RACE analysis. qPCR analysis of liver biopsy specimens demonstrated that these unannotated transcripts, as well as IRF1, TRIM22 and MET, were also upregulated in hepatitis C with mild inflammation and no fibrosis. The analysis of 5′ capped RNA in combination with ENCODE tiling arrays provides additional gene expression information and identifies novel upregulated Pol II transcripts not previously described in HCV infected liver. This approach, particularly when combined with new RNA sequencing technologies, should also be useful in further defining Pol II transcripts differentially regulated in specific disease states and in studying RNAs regulated by changes in pre-mRNA splicing or 3′ polyadenylation status.

The effect of obesity on intrahepatic cytokine and chemokine expression in chronic hepatitis C infection

BackgroundObese subjects with chronic hepatitis C virus (HCV) infection have more rapidly progressive liver disease.ObjectiveIn this study, we aimed to compare the hepatic cytokine and chemokine profiles in obese and...

Hepatitis C virus targets over‐expression of arginase I in hepatocarcinogenesis

Hepatitis C virus (HCV) infection is often associated with chronic liver disease, which is a major risk factor for the development of hepatocellular carcinoma (HCC). To study the HCV host-cell relationship on the molecular level, HepG2 and Huh7 cells were stably transfected with an infectious cDNA clone of HCV or with empty vector. Evidence for HCV replication was obtained in both culture systems. HCV also stimulated growth in vitro. To identify genes whose altered expression by HCV are important to the pathogenesis of infection, RNAs were isolated from HepG2-HCV and HepG2-vector cells and subjected to microarray analysis. The results showed that arginase 1 mRNA and protein were elevated about threefold in HCV positive compared with negative cells (p < 0.01). Arginase 1 expression was elevated in more than 75% of HCV infected liver samples compared with paired HCC from the same patients (>33% positive) and to uninfected liver tissues (0% positive). Arginase 1 specific siRNA inhibited the ability of HCV to stimulate hepatocellular growth in culture by >70%, suggesting that the metabolism of arginine to ornithine may contribute to HCV mediated stimulation of hepatocellular growth. Introduction of arginase specific siRNA also resulted in increased nitric oxide synthase (iNOS) (>1.2-fold), nitric oxide (NO) production (>3-fold) and increased cell death (>2.5-fold) in HCV positive compared with negative cells, suggesting that these molecules potentially contribute to hepatocellular damage. Hence, an important part of the mechanism whereby HCV regulates hepatocellular growth and survival may be through altering arginine metabolism.

Expression of apolipoprotein C-IV is regulated by Ku antigen/peroxisome proliferator-activated receptor γ complex and correlates with liver steatosis

We previously reported that hepatitis C virus (HCV) core protein up regulated transcription of apolipoprotein C-IV (ApoC-IV, 10.7-fold increase), a member of the apolipoprotein family implicated in liver steatosis. Here, we identified host transcription factors regulating the ApoC-IV gene expression. Transcriptional regulators were identified by DNA affinity purification and steatosis was detected by oil red O staining and triglyceride assay. We defined a 163-bp ApoC-IV promoter as a core protein responsive element, and identified Ku antigen complex (Ku70 and Ku80) as well as nuclear receptors PPARgamma/RXRalpha as key regulators of ApoC-IV gene expression. Both Ku70 overexpression and PPARgamma agonist significantly increased ApoC-IV promoter activity; conversely, Ku70 silencing or mutation of PPARgamma binding site diminished the ApoC-IV promoter activity. Interestingly, transient transfection of ApoC-IV cDNA into a human hepatoma cell line was able to trigger moderate lipid accumulation. In agreement with this in vitro study, ApoC-IV transcript level was increased in HCV infected livers which correlated with triglyceride accumulation. ApoC-IV overexpression may perturb lipid metabolism leading to lipid accumulation. HCV core protein may modulate ApoC-IV expression through Ku antigen and PPARgamma/RXRalpha complex.

Advances in Genomic Research on Hepatitis C Virus with a Useful Tool, Replicon System

The research for hepatitis C virus (HCV) has long delayed by missing of in vitro culture system. Since the development of replicon system, a replication system of subgenomic HCV RNAs in a hepatoma cell line, has been reported, many virological and clinical findings have been discovered. Recently, in addition of subgenomic replication system, hepatitis C virus full-length RNA replication has been possible, and a few cell culture systems producing viral particles have been produced. These developments enabled us to investigate the life cycle or intracellular circumstance of HCV production. By screening of newly synthesized drugs with this replicon system, several possible medicines have been established and clinical researches are now running. Among them, VX950 and SCH503034 are nearest to clinical use. Other possible agents for reducing viral replication such as cyclophyllin inhibitors, inhibitors of sphingomyelin synthesis, HMG-CoA reductase inhibitors, or RNA-dependent RNA polymerase inhibitors have been also investigated. Furthermore the mechanism for development of hepatocellular carcinoma in the HCV infected liver has been vigorously studied using the HCV replicon system.

Hepatitis C virus receptor expression in normal and diseased liver tissue

The principal site of hepatitis C virus (HCV) replication is the liver. HCV pseudoparticles infect human liver derived cell lines and this suggests that liver-specific receptors contribute to defining HCV hepatotropism. At least three host cell molecules have been reported to be important for HCV entry: the tetraspanin CD81, scavenger receptor class B member I (SR-BI), and the tight junction (TJ) protein Claudin 1 (CLDN1). Hepatocytes in liver tissue coexpress CD81, SR-BI, and CLDN1, consistent with their ability to support HCV entry. CLDN1 localized at the apical-canalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. In contrast, CLDN1 appeared to colocalize with SR-BI at the basolateral-sinusoidal surface. CLDN1 expression was increased on basolateral hepatocyte membranes in HCV-infected and other chronically inflamed liver tissue compared with normal liver. In contrast, CLDN4 hepatocellular staining was comparable in normal and diseased liver tissue. HCV infection of Huh-7.5 hepatoma cells in vitro significantly increased CLDN1 expression levels, consistent with a direct modulation of CLDN1 by virus infection. In HCV infected livers, immunohistochemical studies revealed focal patterns of CLDN1 staining, suggesting localized areas of increased CLDN1 expression in vivo which may potentiate local viral spread within the liver.

Initial steroid-free immunosuppression after liver transplantation in recipients with hepatitis C virus related cirrhosis.

Steroids can increase hepatitis C virus (HCV) replication. After liver transplantation (LTx), steroids are commonly used for immunosuppression and acute rejection is usually treated by high steroid dosages. Steroids can worsen the outcome of recurrent HCV infection. Therefore, we evaluated the outcome of HCV infected liver recipients receiving initial steroid-free immunosuppression. Thirty patients undergoing LTx received initial steroid-free immunosuppression. Indication for LTx included 7 patients with HCV related cirrhosis. Initial immunosuppression consisted of tacrolimus 2X0.05 mg/kg.d po and mycophenolate mofetil (MMF) 2X15 mg/kg.d po. The tacrolimus dosage was adjusted to trough levels in the target range of 10-15 microg/L during the first 3 mo and 5-10 microg/L thereafter. Manifestations of acute rejection were verified histologically. Patient and graft survival of 30 patients receiving initial steroid-free immunosuppression was 86% and 83% at 1 and 2 years. Acute rejection occurred in 8/30 patients, including 1 HCV infected recipient. All HCV-infected patients had HCV genotype II (1b). HCV seropositivity occurred within the first 4 mo after LTx. The virus load was not remarkably increased during the first year after LTx. Histologically, grafts had no severe recurrent hepatitis. From our experience, initial steroid-free immunosuppression does not increase the risk of acute rejection in HCV infected liver recipients. Furthermore, none of the HCV infected patients developed serious chronic liver diseases. It suggests that it may be beneficial to avoid steroids in this particular group of patients after LTx.

Sustained activation of the Raf/MEK/Erk pathway in response to EGF in stable cell lines expressing the Hepatitis C Virus (HCV) core protein.

Chronic hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and hepatocellular carcinoma (HCC) worldwide. The HCV capside core is a multifunctional protein with regulatory functions that affects transcription and cell growth in vitro and in vivo. Here, we show that both HCV genotype 1a and 3 core proteins activate MEK1 and Erk1/2 MAP kinases and that the costitutive expression of the HCV core results in a high basal activity of Raf1 and MAP/kinase/kinase, as determined by endogenous Raf1 in vitro kinase assay and immunodetection of hyperphosphorylated Erk1 and Erk2 even after a serum starvation. Moreover, the activation of both Erk1/2 and the downstream transcription factor Elk-1 in response to the mitogenic stimulus EGF is significantly prolonged. The sustained response to EGF in cells expressing the HCV core occurs despite a normal induction of the MAP phosphatases MKP regulatory feedback and is likely due to the costitutive activation of Raf-1 activity. The ability of HCV core proteins to directly activate the MAP kinase cascade and to prolong its activity in response to mitogenic stimuli may contribute to the neoplastic transformation of HCV infected liver cells.

Hepatitis C virus infection: immune responsiveness and interferon-alpha treatment.

Hepatitis C virus (HCV) is responsible for most cases of posttransfusion hepatitis and sporadic or community-acquired non-A, non-B hepatitis. Different generations of enzyme-linked immunosorbent assay have been generated for detecting antibodies to HCV epitopes. HCV-RNA quantitative analysis has been developed by means of polymerase chain reaction technique. This approach is the only reliable method for HCV-RNA tissue localization, being helpful in early diagnosis. HCV infected liver is characterized by an inflammatory infiltrate including CD4+, CD8+, and B lymphocytes. Evidence has been provided that in HCV patients CD8+ cell response is associated with low level of viraemia and higher level of disease activity. CD4+ T cells exhibit specificity for the core antigen, also correlating with disease activity and viraemia. Costimulatory molecules, cytokines, oxygen radicals, the complex Fas/Fas-ligand and soluble class I HLA structures are discussed as putative cofactors involved in disease evolution. Various forms of interferon (IFN)-alpha have been evaluated for the treatment of patients with HCV infection. Initial enthusiasm has been attenuated by the evidence of a low sustained virological response rate and the constant side effects of IFN-alpha therapy in patients with chronic HCV disease. Among possible markers for predicting therapeutic outcome in HCV-positive individuals, anti-core antibodies correlate positively with response to IFN-alpha administration, as well as reduction of interleukin-2 serum levels has been detected in patients with a good therapeutic response. Association between HCV infection and autoimmune phenomena, also in relation to IFN-alpha therapy has been reported. Finally, results of the combined treatment with IFN-alpha/ribavirin are illustrated.