Alternative splicing of viral transcripts: the dark side of HBV.

Regulation of alternative splicing is one of the most efficient mechanisms to enlarge the proteomic diversity in eukaryotic organisms. Many viruses hijack the splicing machinery following infection to accomplish their replication cycle. Regarding the HBV, numerous reports have described alternative splicing events of the long viral transcript (pregenomic RNA), which also acts as a template for viral genome replication. Alternative splicing of HBV pregenomic RNAs allows the synthesis of at least 20 spliced variants. In addition, almost all these spliced forms give rise to defective particles, detected in the blood of infected patients. HBV-spliced RNAs have long been unconsidered, probably due to their uneasy detection in comparison to unspliced forms as well as for their dispensable role during viral replication. However, recent data highlighted the relevance of these HBV-spliced variants through (1) the trans-regulation of the alternative splicing of viral transcripts along the course of liver disease; (2) the ability to generate defective particle formation, putative biomarker of the liver disease progression; (3) modulation of viral replication; and (4) their intrinsic propensity to encode for novel viral proteins involved in liver pathogenesis and immune response. Altogether, tricky regulation of HBV alternative splicing may contribute to modulate multiple viral and cellular processes all along the course of HBV-related liver disease.

Hepatitis B virus X protein enhances the development of liver fibrosis and the expression of genes associated with epithelial-mesenchymal transitions and tumor progenitor cells.

The hepatitis B virus X protein (HBx) has pleiotropic biological effects, which underlies its potential role in cell transformation. However, its involvement in hepatic fibrosis remains unclear. In this study, we wanted to clarify, in vivo, the role of HBx protein in the development of liver fibrosis. Mice transgenic for the full-length HBx (FL-HBx) were used. To create liver fibrosis, FL-HBx transgenic and control mice were chronically exposed to carbon tetrachloride (CCl4). Modulation of the expression of proteins involved in matrix remodeling, hepatic metabolism and epithelial-mesenchymal transition (EMT) were investigated. In transgenic mice, FL-HBx expression potentiates CCl4-induced liver fibrosis with increased expression of proteins involved in matrix remodeling (Collagen1a, α-Sma, PdgfR-ß, MMP-13). In FL-HBx transgenic mice, an increase in EMT was observed with a higher transcription of two inflammatory cytokines (TNF-α and TGF-ß) and a decrease of glutamine synthetase expression level. This was associated with a sustained cell cycle and hepatocyte polyploidy alteration consistent with p38 and ERK1/2 overactivation, increase of PLK1 transcription, accumulation of SQSTM1/p62 protein and increase expression of Beclin-1. This correlates with a higher expression of tumor progenitor cell markers (AFP, Ly6D and EpCam), indicating a higher risk of progression from fibrosis to hepatocellular carcinoma (HCC) in the presence of FL-HBx protein. In conclusion, our results show that FL-HBx protein enhances the development of liver fibrosis and contributes to the progression of liver disease from chronic hepatitis to HCC.

Viral Load Affects the Immune Response to HBV in Mice With Humanized Immune System and Liver.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus and how it affects disease progression are unclear. METHODS: We performed studies with BALB/c Rag2-/-Il2rg-/-SirpaNODAlb-uPAtg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (PCR). Plasma levels of HBV DNA were quantified by PCR reaction, and antigen-specific antibodies were detected by immunocytochemistry of HBV-transfected BHK-21 cells. RESULTS: Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e (HBe), core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation. CONCLUSION: In HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.

Alternative splicing of hepatitis B virus: A novel virus/host interaction altering liver immunity.

BACKGROUND & AIMS: Hepatitis B virus (HBV) RNA can undergo alternative splicing, but the relevance of this post-transcriptional regulation remains elusive. The mechanism of HBV alternative splicing regulation and its impact on liver pathogenesis were investigated. METHODS: HBV RNA-interacting proteins were identified by RNA pull-down, combined with mass spectrometry analysis. HBV splicing regulation was investigated in chemically and surgically induced liver damage, in whole HBV genome transgenic mice and in hepatoma cells. Viral and endogenous gene expression were quantified by quantitative reverse transcription polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. Resident liver immune cells were studied by fluorescence-activated cell sorting. RESULTS: HBV pregenomic RNA-interacting proteins were identified and 15% were directly related to the splicing machinery. Expression of these splicing factors was modulated in HBV transgenic mice with liver injuries and contributed to an increase of the HBV spliced RNA encoding for HBV splicing-generated protein (HBSP). HBSP transgenic mice with chemically induced liver fibrosis exhibited attenuated hepatic damage. The protective effect of HBSP resulted from a decrease of inflammatory monocyte/macrophage recruitment through downregulation of C-C motif chemokine ligand 2 (CCL2) expression in hepatocytes. In human hepatoma cells, the ability of HBSP to control CCL2 expression was confirmed and maintained in a whole HBV context. Finally, viral spliced RNA detection related to a decrease of CCL2 expression in the livers of HBV chronic carriers underscored this mechanism. CONCLUSION: The microenvironment, modified by liver injury, increased HBSP RNA expression through splicing factor regulation, which in turn controlled hepatocyte chemokine synthesis. This feedback mechanism provides a novel insight into liver immunopathogenesis during HBV infection. Lay summary: Hepatitis B virus persists for decades in the liver of chronically infected patients. Immune escape is one of the main mechanisms developed by this virus to survive. Our study highlights how the crosstalk between virus and liver infected cells may contribute to this immune escape.

Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-α-stimulated signaling pathways and limits the extent of liver inflammation.

Hepatitis B splicing-regulated protein (HBSP) of the hepatitis B virus (HBV) was uncovered a few years ago but its function remains unknown. HBSP expression occurs from a spliced viral transcript that increases during the course of liver disease. This study aimed at characterizing the impact of HBSP on cellular signaling pathways in vitro and on liver pathogenesis in transgenic (Tg) mice. By RT-qPCR array, NF-κB-inducible genes appeared modulated in HepG2 cells transduced with a HBSP-encoding lentivirus. Using luciferase and Western blot assays, we observed a decreased activation of the NF-κB pathway in HBSP-expressing cells following TNF-α treatment, as illustrated by lower levels of phosphorylated IκB-α. Meanwhile, the level of phosphorylated JNK increased together with the sensitivity to apoptosis. The contrasting effects on JNK and IκB-α activation upon TNF-α stimulation matched with a modulated maturation of TGF-ß-activated kinase 1 (TAK1) kinase, assessed by 2-dimensional SDS-PAGE. Inhibition of the NF-κB pathway by HBSP was confirmed in the liver of HBSP Tg mice and associated with a significant decrease of chemically induced chronic liver inflammation, as assessed by immunohistochemistry. In conclusion, HBSP contributes to limit hepatic inflammation during chronic liver disease and may favor HBV persistence by evading immune response.

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.

Regulation of interleukin-6 in head and neck squamous cell carcinoma is related to papillomavirus infection.

The prevalence of head and neck squamous cell carcinoma (HNSCC) related to human papillomavirus (HPV) is increasing, unlike tobacco- and alcohol-associated cancers. To gain a clearer understanding of the molecular mechanisms implicated in HNSCC, depending on the presence or not of a viral sequence, we investigated the expression of proteins detected in the tumor regions of HNSCC patients. Twenty-two untreated HNSCC patients were selected according to the presence of HPV-16. For six patients, tumor and controlateral healthy tissues were tested for viral detection before quantitative proteomic analysis. After confirmation by Western blot, proteins were connected into a network, leading to investigate interleukin-6 (IL-6) by immunocytochemistry and ELISA. 41 ± 5% of proteins quantified by proteomics were differentially expressed in tumor compared with healthy regions. Among them, 36 proteins were retained as modulated in HPV-16 positive or negative tumors, including cytokeratins, tubulins, annexin A1, and serpin B1. Network analysis suggested a central role of IL-6, confirmed by overexpression of IL-6 in tumor tissues as in sera of HPV-negative HNSCC compared with HPV-16-positive tumors. This modulation may contribute to the survival and proliferation of cancer cells, although it was not related to tumor stage or to the level of HPV-16 DNA.

IL-15 transpresentation promotes both human T-cell reconstitution and T-cell-dependent antibody responses in vivo.

Cytokine immunotherapies targeting T lymphocytes are attractive clinical interventions against viruses and tumors. In the mouse, the homeostasis of memory α/ß CD8(+) T cells and natural killer (NK) cells is significantly improved with increased IL-15 bioavailability. In contrast, the role of "transpresented" IL-15 on human T-cell development and homeostasis in vivo is unknown. We found that both CD8 and CD4 T cells in human immune system (HIS) mice are highly sensitive to transpresented IL-15 in vivo, with both naïve (CD62L(+)CD45RA(+)) and memory phenotype (CD62L(-)CD45RO(+)) subsets being significantly increased following IL-15 "boosting." The unexpected global improvement in human T-cell homeostasis involved enhanced proliferation and survival of both naïve and memory phenotype peripheral T cells, which potentiated B-cell responses by increasing the frequency of antigen-specific responses following immunization. Transpresented IL-15 did not modify T-cell activation patterns or alter the global T-cell receptor (TCR) repertoire diversity. Our results indicate an unexpected effect of IL-15 on human T cells in vivo, in particular on CD4(+) T cells. As IL-15 promotes human peripheral T-cell homeostasis and increases the frequency of neutralizing antibody responses in HIS mice, IL-15 immunotherapy could be envisaged as a unique approach to improve vaccine responses in the clinical setting.

La protéine X du virus de l'hépatite B et son rôle dans le développement du carcinome hépatocellulaire.

Hepatocellular carcinoma (HCC) is the most frequent form of liver cancer worldwide, and represents the third cause of death. While epidemiological studies have clearly established that hepatitis B virus (HBV) infection is a major risk factor for the development of HCC, the molecular mechanisms underlying virally-induced tumourigenesis are not fully understood. The transcriptional regulatory HBx protein has been described as a multifunctional protein exhibiting numerous activities affecting gene transcription, intracellular signal transduction, cell proliferation, apoptosis and DNA repair. While any or all of the multiple activities of HBx could contribute to hepato-carcinogenesis, HBx is not considered as an oncogene. HBx rather acts as a co-factor of carcinogenesis, through the up-regulation of a large number of cellular genes involved in oncogenesis, proliferation, inflammation and immune response. In this review, we will summarize the current knowledge on the mechanisms involving HBx protein in liver carcinogenesis.

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.

Hepatitis delta virus proteins repress hepatitis B virus enhancers and activate the alpha/beta interferon-inducible MxA gene.

Co-infection and superinfection of hepatitis B virus (HBV) with hepatitis delta virus (HDV) leads to suppression of HBV replication both in patients and in animal and cellular models. The mechanisms behind this inhibition have not previously been explored fully. HBV replication is governed by four promoters and two enhancers, Enh1 and Enh2. Repression of these enhancers has been reported to be one of the main mechanisms of HBV inhibition. Moreover, in a previous study, it has been demonstrated that alpha interferon (IFN-alpha)-inducible MxA protein inhibits HBV replication. HDV encodes two proteins, p24 and p27. p27 was shown to activate several heterologous promoters, including HBV promoters. In an attempt to analyse the mechanisms of HBV inhibition by HDV, the question was raised whether HDV proteins could act directly by repressing HBV enhancers, and/or indirectly by activating the MxA gene. This issue was addressed in a co-transfection model in Huh-7 cells, using p24- or p27-expressing plasmids along with Enh1, Enh2, HBV and MxA promoter-luciferase constructs. Enh1 and Enh2 were strongly repressed, by 60 and 80 % and 40 and 60 %, by p24 and p27, respectively. In addition, p27 was responsible for threefold activation of the MxA promoter and potentiation of IFN-alpha on this promoter. MxA mRNA quantification and a virus yield reduction assay confirmed these results. In conclusion, this study shows that HDV proteins inhibit HBV replication by trans-repressing its enhancers and by trans-activating the IFN-alpha-inducible MxA gene.

Hepatitis B virus X protein promotes DNA damage propagation through disruption of liver polyploidization and enhances hepatocellular carcinoma initiation.

Hepatitis B virus X protein (HBx) contributes to Hepatitis B virus (HBV)-related liver cancer. However, its impact on hepatocyte proliferation and genomic stability remains elusive. We studied the role of HBx expression on the progression of cell cycle and liver polyploidization during proliferation and liver carcinogenesis. Full-length HBx transgenic mice (FL-HBx) were developed to investigate liver ploidy as well as hepatocyte proliferation, along normal liver maturation and during cancer initiation (chemical carcinogen treatment). Investigation of postnatal liver development in FL-HBx showed an aberrant G1/S and G2/M transitions, triggered (1) a delay of the formation of hepatocytes binucleation, (2) the early synthesis of polyploidy nuclei (≥4n) and (3) DNA damage appearance. Moreover, HBV infection during hepatocytes proliferation in a humanized liver mouse model led, to modifications in polyploidy of hepatocytes. In initiation of hepatocellular carcinoma, FL-HBx protein decreased ChK1 phosphorylation, Mre11 and Rad51 expression, upregulated IL-6 expression and impaired apoptosis. This was related to DNA damage accumulation in FL-HBx mice. At day 75 after initiation of hepatocellular carcinoma, FL-HBx mice revealed significant cell cycle changes related to the increased amount of 4n nuclei and of markers of cancer progenitor cells. Finally, PLK1 upregulation and p38/ERK activation in FL-HBx mice were implicated in aberrant polyploidization favoring DNA damage propagation and hepatocyte transformation. In conclusion, our data indicate that FL-HBx protein increases DNA damage through the hijack of hepatocyte polyploidization. That leads to enhancement of hepatocellular carcinoma initiation in an inflammatory context.

Alteration of splicing factors' expression during liver disease progression: impact on hepatocellular carcinoma outcome.

PURPOSE: Trans-acting splicing factors (SF) shape the eukaryotic transcriptome by regulating alternative splicing (AS). This process is recurrently modulated in liver cancer suggesting its direct contribution to the course of liver disease. The aim of our study was to investigate the relationship between the regulation of SFs expression and liver damage. METHODS: The expression profile of 10 liver-specific SF and the AS events of 7 genes associated with liver disorders was assessed by western-blotting in 6 murine models representing different stages of liver damage, from inflammation to hepatocellular carcinoma (HCC). Relevant SFs (PSF, SRSF3, and SRSF6) and target genes (INSR, SRSF3, and SLK) modulated in mice were investigated in a cohort of 179 HCC patients. RESULTS: Each murine model of liver disease was characterized by a unique SF expression profile. Changes in the SF profile did not affect AS events of the selected genes despite the presence of corresponding splicing sites. In human HCC expression of SFs, including the tumor-suppressor SRSF3, and AS regulation of genes studied were frequently upregulated in tumor versus non-tumor tissues. Risk of tumor recurrence positively correlated with AS isoform of the INSR gene. In contrast, increased levels of SFs expression correlated with an extended overall survival of patients. CONCLUSIONS: Dysregulation of SF expression is an early event occurring during liver injury and not just at the stage of HCC. Besides impacting on AS regulation, overexpression of SF may contribute to preserving hepatocyte homeostasis during liver pathogenesis.

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.

Prospective comparison of Abbott RealTime HBV DNA and Versant HBV DNA 3.0 assays for hepatitis B DNA quantitation: impact on HBV genotype monitoring.

The quantitation of human hepatitis B virus (HBV) in the serum of infected patients is recommended to characterize the course of chronic HBV infection. The aim of this prospective study was to evaluate the performance of the Abbott RealTime PCR assay for HBV DNA quantitation by comparison with the standard Versant HBV DNA 3.0 assay. The better sensitivity and broader dynamic range of HBV DNA quantitation using the Abbott RealTime PCR assay was confirmed by the study of 362 serum samples from 311 patients. In addition, data analysis revealed the concordance of HBV DNA quantitations between the two assays. When this evaluation was assessed as a function of HBV genotype, there was discordance for HBV genotype C samples. Thus, we performed an in-house PCR to confirm the discrepancy observed regarding the HBV genotypes. The in-house PCR results agreed better with the Abbott RealTime PCR method when compared with the standard hybridization assay. In conclusion, the wide dynamic range of HBV DNA quantitation achieved with the Abbott RealTime PCR assay makes it appropriate for the clinical monitoring of HBV infected patients. However, a change of HBV DNA quantitation method could influence results on the follow-up of HBV genotype C infected patients.

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.

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.

Modeling hepatitis virus infections and treatment strategies in humanized mice.

Hepatitis viruses cause chronic liver diseases such as fibrosis, cirrhosis and hepatocellular carcinomas that are difficult to treat and constitute a global health problem. Species-specific viral tropism has limited the usefulness of small animal models to study the impact of viral hepatitis. Immunodeficient mice grafted with human hepatocytes are susceptible to hepatitis viruses B, C, D and E (HBV, HCV, HDV and HEV), developing full viral life cycles, and delivering a means to investigate virus-host interactions and antiviral treatments. These chimeric humanized mouse models have been further grafted with humanized immune systems to decipher immune responses following hepatotropic viral infections, the ensuing pathophysiology, and to test novel therapeutic strategies.

Identification, using cDNA macroarray analysis, of distinct gene expression profiles associated with pathological and virological features of hepatocellular carcinoma.

It is still unclear as to whether the gene expression profile in HCV- or HBV-related HCC exhibits a degree of specificity and whether the development of HCC in a context of cirrhosis influences this gene profile. To address these issues, the expression profiles of 15 cases of HCC were analysed using cDNA macroarray. A global analysis and hierarchical clustering, demonstrated the heterogeneity of HCC patterns, with a majority of down-regulated genes. Statistical analysis clearly showed a distinction between the gene expression profiles of HCV- and HBV-related HCC. HBV-associated HCC exhibited involvement of different cellular pathways, those controlling apoptosis, p53 signalling and G1/S transition. In HCV-related HCC we identified a more heterogenous pattern with an over-expression of the TGF-beta induced gene. In HCC developing on non-cirrhotic tissues, beta-catenin encoding gene and genes implicated in the PKC pathway were specifically up-regulated. In addition, our investigation highlighted a distinct profiles of TGF-beta superfamily encoding genes in well, moderately or poorly differentiated HCC. Overall, our study supports the hypothesis that despite the heterogeneity of the HCC pattern, the large-scale screening of gene expression may provide data significant to our understanding of the mechanism of liver carcinogenesis.

A novel mouse model for stable engraftment of a human immune system and human hepatocytes.

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20-50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.