Haizhen Zhu

Cyclosporine suppresses hepatitis C virus in vitro and increases the chance of a sustained virological response after liver transplantation.

Cyclosporine is an immunosuppressive agent widely used in the management of liver transplant recipients. Cyclosporine has been shown to have antiviral activities against HIV, herpes simplex, and vaccinia viruses. The aim of this study was to determine the effect of Cyclosporine in viral clearance in the liver transplant recipients during therapy with combination of interferon and ribavirin, and to determine the anti‐viral potential of Cyclosporine in vitro. Immunosuppression consisted of either Cyclosporine or Tacrolimus‐based therapy. Both groups received therapy with interferon and ribavirin for 48 weeks when evidence of progressive histologic disease was determined. We found that subjects on Cyclosporine‐based immunosuppression (n = 56) had a higher sustained virological response of 46% compared to 27% in the patients on Tacrolimus‐based therapy (n=59, P = 0.03). In vitro studies were performed to evaluate the antiviral effect of Cyclosporine in the replicon system. These studies showed that Cyclosporine inhibits hepatitis C viral replication in a dose‐dependent manner. Combination of Cyclosporine with interferon showed additive effect, and its function is independent of interferon signaling pathways. In conclusion, Cyclosporine may offer an advantage to Tacrolimus in those patients undergoing interferon‐based therapy and should be studied in a prospective randomized trial. Liver Transpl 12:51–57, 2006.

The antigen for Hep Par 1 antibody is the urea cycle enzyme carbamoyl phosphate synthetase 1

Hepatocyte paraffin 1 (Hep Par 1), a murine monoclonal antibody, is widely used in surgical pathology practice to determine the hepatocellular origin of neoplasms. However, identity of the antigen for Hep Par 1 is unknown. The aim of this study was to characterize the Hep Par 1 antigen. To identify the antigen, immunoprecipitation was used to isolate the protein from human liver tissue, and a distinct protein band was detected at approximately 165 kDa. The protein band was also present in small intestinal tissue, but was not present in several other non-liver tissues nor in three human hepatocellular carcinoma cell lines, Huh-7, HepG2, and LH86. The protein was purified and analyzed by mass spectrometry. It was identified as carbamoyl phosphate synthetase 1 (CPS1). CPS1 is a rate-limiting enzyme in urea cycle and is located in mitochondria. We demonstrated that hepatoid tumors (gastric and yolk sac) were immunoreactive with both Hep Par 1 antibody and anti-CPS1 antibody, further confirming the results of mass spectrometric analysis. We found that the three human hepatocellular carcinoma cell lines do not express either CPS1 RNA or protein. We confirmed that the gene was present in these cell lines, suggesting that suppression of CPS1 expression occurs at the transcriptional level. This finding may have relevance to liver carcinogenesis, since poorly differentiated hepatocellular carcinomas exhibit poor to absent immunoreactivity to Hep Par 1. In conclusion, we have identified the antigen for Hep Par 1 antibody as a urea cycle enzyme CPS1. Our results should encourage further investigation of potential role that CPS1 expression plays in liver pathobiology and carcinogenesis.

Novel type I interferon IL-28A suppresses hepatitis C viral RNA replication

Interferon alpha (IFN-α)-based therapy is the currently approved treatment for chronic hepatitis C viral infection. The sustained antiviral response rate is approximately 50% for genotype-1 infection. The major challenge to the HCV community is to improve antiviral efficacy and to reduce the side effects typically seen in IFNα-based therapy. One of the strategies is to identify new interferons, which may have better efficacy and less undesirable side effects. In this report, we examined the role of IL-28A (IFN λ2), a novel type I IFN, in suppression of human hepatitis C viral RNA replication. We have cloned both the human genomic DNA and cDNA of IL-28A, and evaluated their biological activity using HCV RNA replicon cell culture system. The results show that IL-28A effectively inhibits HCV subgenomic RNA replication in a dose-dependent manner. Treatment of human hepatoma cells with IL-28A activates the JAK-STAT signaling pathway and induces the expression of some interferon-stimulated genes (ISGs), such as 6–16 and 1–8U. We also demonstrate that IL-28A induces expression of HLA class I antigens in human hepatoma cells. Moreover, IL-28A appears to specifically suppress HCV IRES-mediated translation. Although IL-28A receptor shares one subunit with the IL-10 receptor, IL-10 treatment has no detectable effect on IL-28A-induced antiviral activity. Interestingly, IL-28A can synergistically enhance IFNα antiviral efficacy. Our results suggest that IL-28A antiviral activity is associated with the activation of the JAK-STAT signaling pathway and expression of ISGs. The effectiveness of IL-28A antiviral activity and its synergistic effect on IFN-α indicate that IL-28A may be potentially used to treat HCV chronic infection.

Hepatocellular carcinoma cell supernatants increase expansion and function of CD4(+)CD25(+) regulatory T cells.

Dysfunction of the host immune system in cancer patients can be due to a number of factors, including suppression of tumor-associated antigen reactive lymphocytes by CD4+CD25+ regulatory T (Treg) cells. Several studies suggest that Tregs are elevated in cancer patients and that depletion of Tregs may enhance the antitumor immunity of host, but the pathogenic and mechanistic relationship between cancer and Tregs is still unclear. In this report, we show that Tregs are increased in peripheral blood mononuclear cells (PBMCs) from hepatocellular carcinoma (HCC) patients and positively correlate with tumor burden. When PBMCs are co-cultured with human hepatoma cell lines Huh7, HepG2, and Hclone5, CD4+CD25+-T cell populations increase in frequency and undergo phenotypic and functional changes. CD45RA, CD45RO, CD69, CD62L, GITR, CTLA-4, Ki67, granzyme A, granzyme B, and FOXP3 expression were upregulated in CD4+CD25+ cells after in vitro exposure to HCC cell lines. CD4+CD25+ T cells from PBMCs that were co-cultured with Huh7 cells also have higher suppressor ability compared to that of the CD4+CD25+ T cells from control PBMC. Huh7 culture supernatants appear to promote CD4+CD25+ T-cell proliferation and inhibit CD4+CD25− T-cell proliferation. In conclusion, these results strongly suggest that tumor-related factors not only induce and expand CD4+CD25+ cells, but also enhance their suppressor ability.

Complete replication of hepatitis B virus and hepatitis C virus in a newly developed hepatoma cell line

Significance More than 500 million people are persistently infected with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) and are at a risk of developing chronic hepatitis, cirrhosis, and liver cancer. The absence of robust cell culture systems for both viral infections limits the understanding of the virus lifecycle and pathogenesis required for the development of vaccine and antivirals. We have established a novel human hepatoma cell line termed “HLCZ01” that supports the entire lifecycle of both HBV and HCV produced both in cell culture and clinically. This cell line provides a powerful tool for addressing the virus lifecycle and the development of antivirals and vaccines. The absence of a robust cell culture system for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has limited the analysis of the virus lifecycle and drug discovery. We have established a hepatoma cell line, HLCZ01, the first cell line, to the authors’ knowledge, supporting the entire lifecycle of both HBV and HCV. HBV surface antigen (HBsAg)-positive particles can be observed in the supernatant and the lumen of the endoplasmic reticulum of the cells via electron microscopy. Interestingly, HBV and HCV clinical isolates propagate in HLCZ01 cells. Both viruses replicate in the cells without evidence of overt interference. HBV and HCV entry are blocked by antibodies against HBsAg and human CD81, respectively, and the replication of HBV and HCV is inhibited by antivirals. HLCZ01 cells mount an innate immune response to virus infection. The cell line provides a powerful tool for exploring the mechanisms of virus entry and replication and the interaction between host and virus, facilitating the development of novel antiviral agents and vaccines.

Stabilized β-catenin promotes hepatocyte proliferation and inhibits TNFα-induced apoptosis

The human hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The mechanisms of liver cell oncogenic transformation are still unknown. The β-catenin mutations are identified in up to 30% of HCC and 80% of hepatoblastoma, suggesting a potential role of β-catenin in the pathogenesis of liver cancers. To define the biological role of the stabilized β-catenin in liver cell growth and transformation, we examined the effect of mutant β-catenin on an immortalized murine hepatocyte cell line, AML12. A cell line that stably expresses mutant β-catenin was established. The cell proliferation, apoptosis, and cell transformation of this cell line were characterized. Our data indicate that the stabilized β-catenin enhances hepatocyte proliferation, suppresses TNFα/Act D-induced cell apoptosis, and causes weak anchorage-independent cell growth. The stabilized β-catenin-containing cells did not develop tumor in immune-deficient mice. The target genes, c-myc and cyclin D1, were activated by β-catenin in the hepatocytes. Our study suggests that mutant β-catenin can promote cell proliferation and cell survival ability, but the stabilized β-catenin alone is insufficient for completely oncogenic transformation.

Anti‐hepatitis C virus activity of albinterferon alfa‐2b in cell culture

Background:  Interferon‐based combination therapy is the standard treatment for chronic hepatitis C virus (HCV) infection. The weekly administration of long‐acting pegylated interferons (PEG‐IFNα‐2a or PEG‐IFNα‐2b) provides superior antiviral efficacy over standard interferon alfa (IFNα) for the treatment of HCV infection. Albinterferon alfa‐2b (alb‐IFN) is a novel recombinant protein consisting of IFNα‐2b that is genetically fused to human albumin.

Inhibition of Hepatitis C Virus Infection by DNA Aptamer against Envelope Protein

ABSTRACT Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-β) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients.

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

ABSTRACT Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C.

MiR-942 Mediates Hepatitis C Virus-Induced Apoptosis via Regulation of ISG12a

The interaction between hepatitis C virus (HCV) and human hepatic innate antiviral responses is unclear. The aim of this study was to examine how human hepatocytes respond to HCV infection. An infectious HCV isolate, JFH1, was used to infect a newly established human hepatoma cell line HLCZ01. Viral RNA or NS5A protein was examined by real-time PCR or immunofluorescence respectively. The mechanisms of HCV-induced IFN-β and apoptosis were explored. Our data showed that HLCZ01 cells supported the entire HCV lifecycle and IFN-β and interferon-stimulated genes (ISGs) were induced in HCV-infected cells. Viral infection caused apoptosis of HLCZ01 cells. Silencing of RIG-I, IRF3 or TRAIL inhibited ISG12a expression and blocked apoptosis of viral-infected HLCZ01 cells. Knockdown ISG12a blocked apoptosis of viral-infected cells. MiR-942 is a candidate negative regulator of ISG12a predicted by bioinformatics search. Moreover, HCV infection decreased miR-942 expression in HLCZ01 cells and miR-942 was inversely correlated with ISG12a expression in both HCV-infected cells and liver biopsies. MiR-942 forced expression in HLCZ01 cells decreased ISG12a expression and subsequently suppressed apoptosis triggered by HCV infection. Conversely, silencing of miR-942 expression by anti-miR-942 increased ISG12a expression and enhanced apoptosis in HCV-infected cells. Induction of Noxa by HCV infection contributed to ISG12a-mediated apoptosis. All the data indicated that innate host response is intact in HCV-infected hepatocytes. MiR-942 regulates HCV-induced apoptosis of human hepatocytes by targeting ISG12a. Our study provides a novel mechanism by which human hepatocytes respond to HCV infection.