C-terminal-truncated hepatitis B virus X protein enhances the development of diethylnitrosamine-induced hepatocellular carcinogenesis.

Hepatitis B virus X protein (HBx) is involved in the development of hepatocellular carcinoma (HCC). The HBx sequence is a preferential site of integration into the human genome, leading to the formation of C-terminal-truncated HBx proteins (Ct-HBx). We previously reported that Ct-HBx proteins were able to potentiate cell transformation in vitro. Our present goal was to compare the ability of Ct-HBx and full-length HBx (FL-HBx) proteins to develop or enhance HCC in transgenic mice. In the absence of treatment, neither Ct-HBx- nor FL-HBx-transgenic mice developed HCC. In young mice treated with diethylnitrosamine (DEN) at 8 months of age, a significantly higher incidence and number of liver lesions were observed in Ct-HBx mice than in FL-HBx and control mice. The earlier development of tumours in Ct-HBx-transgenic mice was associated with increased liver inflammation. At 10 months, macroscopic and microscopic analyses showed that, statistically, FL-HBx mice developed more liver lesions with a larger surface area than control mice. Furthermore, during DEN-induced initiation of HCC, Ct-HBx- and FL-HBx-transgenic mice showed higher expression of IL-6, TNF-α and IL-1ß transcripts, activation of STAT3, ERK and JNK proteins and an increase in cell apoptosis. In conclusion, in DEN-treated transgenic mice, the expression of Ct-HBx protein causes a more rapid onset of HCC than does FL-HBx protein. HBV genome integration leading to the expression of a truncated form of HBx protein may therefore facilitate HCC development in chronically infected patients.

Hepatitis B virus HBx protein impairs liver regeneration through enhanced expression of IL-6 in transgenic mice.

BACKGROUND & AIMS: Conflicting results have been reported regarding the impact of hepatitis B virus X protein (HBx) expression on liver regeneration triggered by partial hepatectomy (PH). In the present report we investigated the mechanisms by which HBx protein alters hepatocyte proliferation after PH. METHODS: PH was performed on a transgenic mouse model in which HBx expression is under the control of viral regulatory elements and liver regeneration was monitored. LPS, IL-6 neutralizing antibody, and SB203580 were injected after PH to evaluate IL-6 participation during liver regeneration. RESULTS: Cell cycle progression of hepatocytes was delayed in HBx transgenic mice compared to WT animals. Moreover, HBx induced higher secretion of IL-6 soon after PH. Upregulation of IL-6 was associated with an elevation of STAT3 phosphorylation, SOCS3 transcript accumulation and a decrease in ERK1/2 phosphorylation in the livers of HBx transgenic mice. The involvement of IL-6 overexpression in cell cycle deregulation was confirmed by the inhibition of liver regeneration in control mice after the upregulation of IL-6 expression using LPS. In addition, IL-6 neutralization with antibodies was able to restore liver regeneration in HBx mice. Finally, the direct role of p38 in IL-6 secretion after PH was demonstrated using SB203580, a pharmacological inhibitor. CONCLUSIONS: HBx is able to induce delayed hepatocyte proliferation after PH, and HBx-induced IL-6 overexpression is involved in delayed liver regeneration. By modulating IL-6 expression during liver proliferation induced by stimulation of the cellular microenvironment, HBx may participate in cell cycle deregulation and progression of liver disease.

Antiviral activity of Bay 41-4109 on hepatitis B virus in humanized Alb-uPA/SCID mice.

Current treatments for HBV chronic carriers using interferon alpha or nucleoside analogues are not effective in all patients and may induce the emergence of HBV resistant strains. Bay 41-4109, a member of the heteroaryldihydropyrimidine family, inhibits HBV replication by destabilizing capsid assembly. The aim of this study was to determine the antiviral effect of Bay 41-4109 in a mouse model with humanized liver and the spread of active HBV. Antiviral assays of Bay 41-4109 on HepG2.2.15 cells constitutively expressing HBV, displayed an IC(50) of about 202 nM with no cell toxicity. Alb-uPA/SCID mice were transplanted with human hepatocytes and infected with HBV. Ten days post-infection, the mice were treated with Bay 41-4109 for five days. During the 30 days of follow-up, the HBV load was evaluated by quantitative PCR. At the end of treatment, decreased HBV viremia of about 1 log(10) copies/ml was observed. By contrast, increased HBV viremia of about 0.5 log(10) copies/ml was measured in the control group. Five days after the end of treatment, a rebound of HBV viremia occurred in the treated group. Furthermore, 15 days after treatment discontinuation, a similar expression of the viral capsid was evidenced in liver biopsies. Our findings demonstrate that Bay 41-4109 displayed antiviral properties against HBV in humanized Alb-uPA/SCID mice and confirm the usefulness of Alb-uPA/SCID mice for the evaluation of pharmaceutical compounds. The administration of Bay 41-4109 may constitute a new strategy for the treatment of patients in escape from standard antiviral therapy.

Inhibition of hepatitis B virus DNA replication by a thermostable interferon-γ variant.

BACKGROUND: Treatment of HBV chronic carriers using interferon (IFN)-α or nucleoside/nucleotide analogues fails to suppress viral infection. Type-II IFN-γ has been shown to inhibit HBV replication. The goal of the present work was to evaluate the antiviral efficacy against HBV of a thermostable IFN-γ variant isolated using Massive Mutagenesis and thermoresistant selection (THR) technologies. METHODS: The thermostability of wild-type (wt) and S63C IFN-γ was determined in vitro and in vivo. Activation of the IFN-γ responsive element by wt and S63C IFN-γ was tested using a luciferase assay. HepG2.2.15 cells constitutively expressing HBV were used to analyse the antiviral activity of wt and S63C IFN-γ against HBV replication. Intracellular HBV DNA was detected by Southern blot and quantified by real-time PCR analyses. RESULTS: S63C IFN-γ was shown to be more thermostable and had a longer half-life than wt IFN-γ. Both wt and S63C IFN-γ displayed a similar capacity to activate the IFN pathway. The treatment of HepG2.2.15 cells with wt or S63C IFN-γ induced the inhibition of HBV viral replication. After heating, S63C IFN-γ displayed better conservation of its antiviral activity against HBV when compared with wt IFN-γ. CONCLUSIONS: These results confirm that the THR method can be used to isolate mutants with enhanced thermostability and demonstrate that a thermostable IFN-γ variant presents antiviral properties against HBV replication. This molecule could provide a new strategy to treat patients who do not respond to antiviral therapy.

Humanized mice for modeling human infectious disease: challenges, progress, and outlook.

Over 800 million people worldwide are infected with hepatitis viruses, human immunodeficiency virus (HIV), and malaria, resulting in more than 5 million deaths annually. Here we discuss the potential and challenges of humanized mouse models for developing effective and affordable therapies and vaccines, which are desperately needed to combat these diseases.

Rescue of fertility in homozygous mice for the urokinase plasminogen activator transgene by the transplantation of mouse hepatocytes.

Development of the urokinase plasminogen activator/SCID (uPA/SCID) transgenic mouse model has opened new perspectives for the study of different biological mechanisms such as liver regeneration, stem cell differentiation, and human hepatic pathogens. We observed that homozygous uPA/SCID mice (uPA+/+/SCID) had a small offspring, indicating a fertility defect. The goal of this study was thus to rescue the fertility of homozygous uPA mice. A deregulation of ovarian function with an absence of corpus luteum was observed in female uPA+/+/SCID mice. In male uPA+/+/SCID mice, a decrease of the weight of the testes, epididymis, seminal vesicle, and prostate was measured. This was associated with an absence of seminal and prostatic secretions and a reduction in testicular sperm production. We hypothesized that the infertility of mice was the consequence of uPA-induced liver injury. Thus, in order to rescue liver function, hepatocytes from mice negative for the uPA transgene were transplanted into uPA+/+/SCID mice. Thirty days after cell transplantation, the livers of transplanted uPA+/+/SCID mice were totally repopulated and presented a normal morphology. Furthermore, transplantation restored normal body weight, life span, and reproductive organ function. In conclusion, we demonstrated that the transplantation of uPA+/+/SCID mice with healthy hepatocytes was sufficient to rescue the reproductive capacity of female and male uPA homozygous animals, highlighting the importance of normal liver function to reproductive capability.

Cell therapy for the diseased liver: from stem cell biology to novel models for hepatotropic human pathogens.

It has long been known that hepatocytes possess the potential to replicate through many cell generations because regeneration can be achieved in rodents after serial two-thirds hepatectomy. It has taken considerable time and effort to harness this potential, with liver regeneration models involving hepatocyte transplantation developing over the past 15 years. This review will describe the experiments that have established the models and methodology for liver repopulation, and the use of cells other than adult hepatocytes in liver repopulation, including hepatic cell lines and hematopoietic, cord blood, hepatic and embryonic stem cells. Emphasis will be placed on the characteristics of the models and how they can influence the outcome of the experiments. Finally, an account of the development of murine models that are competent to accept human hepatocytes is provided. In these models, liver deficiencies are induced in immunodeficient mice, where healthy human cells have a selective advantage. These mice with humanized livers provide a powerful new experimental tool for the study of human hepatotropic pathogens.

Gene modulation associated with inhibition of liver regeneration in hepatitis B virus X transgenic mice.

AIM: To analyze the modulation of gene expression profile associated with inhibition of liver regeneration in hepatitis B X (HBx)-expressing transgenic mice. METHODS: Microarray technology was performed on liver tissue obtained from 4 control (LacZ) and 4 transgenic mice (HBx-LacZ), 48 h after partial hepatectomy. The significance of the normalized log-ratios was assessed for each gene, using robust t-tests under an empirical Bayes approach. Microarray hybridization data was verified on selected genes by quantitative PCR. RESULTS: The comparison of gene expression patterns showed a consistent modulation of the expression of 26 genes, most of which are implicated in liver regeneration. Up-regulated genes included DNA repair proteins (Rad-52, MSH6) and transmembrane proteins (syndecan 4, tetraspanin), while down-regulated genes were connected to the regulation of transcription (histone deacetylase, Zfp90, MyoD1) and were involved in the cholesterol metabolic pathway and isoprenoid biosynthesis (farnesyl diphosphate synthase, Cyp7b1, geranylgeranyl diphosphate synthase, SAA3). CONCLUSION: Our results provide a novel insight into the biological activities of HBx, implicated in the inhibition of liver regeneration.

Transplanted hepatocytes over-expressing FoxM1B efficiently repopulate chronically injured mouse liver independent of donor age.

Orthotopic liver transplantation is limited by the shortage of liver donors, leading to elderly patients being enrolled as donors with increasing frequency. Alternative strategies such as cell therapy are therefore needed. Because transplanted hepatocytes do not proliferate into a recipient liver, repopulation strategies have been developed. We have previously published a proof of concept that hepatocytes harboring a survival selective advantage can efficiently repopulate a mouse liver. We develop here an alternative approach by conferring a selective proliferative advantage on transplanted hepatocytes over resident ones. FoxM1B is a transcription factor that, when over-expressed into hepatocytes, accelerates the cell cycle and maintains the hepatocyte in vivo proliferative capacity of aged livers. We now demonstrate that transplanted hepatocytes over-expressing FoxM1B repopulate the liver of mice subjected to continuous injury far more efficiently than control hepatocytes. We show that old hepatocytes that over-express FoxM1B retain their cell division capacity and repopulate liver as well as young ones, in contrast with old non-modified hepatocytes, which lose their proliferative capacity. In conclusion, our results point to the potential use of FoxM1B expression in hepatocyte-based therapy protocols in diseases where host hepatocytes are chronically injured, especially if donor hepatocytes come from old livers.

New animal models for hepatitis C viral infection and pathogenesis studies.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC). In man, the pathobiological changes associated with HCV infection have been attributed to both the immune system and direct viral cytopathic effects. Until now, the lack of simple culture systems to infect and propagate the virus has hampered progress in understanding the viral life cycle and pathogenesis of HCV infection, including the molecular mechanisms implicated in HCV-induced HCC. This clearly demonstrates the need to develop small animal models for the study of HCV-associated pathogenesis. This review describes and discusses the development of new HCV animal models to study viral infection and investigate the direct effects of viral protein expression on liver disease.