Enhanced B-cell differentiation and reduced proliferative capacity in chronic hepatitis C and chronic hepatitis B virus infections.

BACKGROUND & AIMS: Chronic microbial infections are frequently associated with B-cell activation and polyclonal proliferation, potentially leading to autoimmunity and lymphoproliferative disorders. We assessed B-cell phenotype and function in chronic hepatitis B (HBV) and chronic hepatitis C (HCV) virus infection. METHODS: We studied 70 patients with chronic HCV infection, 34 with chronic HBV infection and 54 healthy controls. B-cell phenotype was assessed by flow cytometry using monoclonal antibodies specific for CD27, the CD69, CD71, and CD86 activation markers and the chemokine receptor CXCR3. Differentiation into immunoglobulin-producing cells (IPC) was analysed by ELISpot upon stimulation and with CD40 ligand±IL-10 as surrogate bystander T-cell help or CpG oligodeoxynucleotide±IL-2, as innate immunity signal. Proliferation was examined by flow cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE) after stimulation with CpG. RESULTS: A significantly higher proportion of B cells from both HCV- and HBV-infected patients expressed activation markers compared with controls and a positive correlation was found between CXCR3(+) B cells and HCV RNA values. Memory B cells from patients with chronic HCV and HBV infections showed enhanced differentiation into IPC compared with controls, although this was restricted to IgG and at a lower level in HCV-compared with HBV-infected patients. Moreover, patients' activated B cells displayed significantly lower proliferative ability compared to healthy donors despite low expression of the FcRL4 exhaustion marker. CONCLUSIONS: B-cell activation, but not exhaustion, is common in chronic viral hepatitis. However, enhanced B-cell differentiation and deficient proliferative capacity were not associated with commitment to terminal differentiation.

Systemic and intrahepatic interferon-gamma-inducible protein 10 kDa predicts the first-phase decline in hepatitis C virus RNA and overall viral response to therapy in chronic hepatitis C.

UNLABELLED: High systemic levels of interferon-gamma-inducible protein 10 kDa (IP-10) at onset of combination therapy for chronic hepatitis C virus (HCV) infection predict poor outcome, but details regarding the impact of IP-10 on the reduction of HCV RNA during therapy remain unclear. In the present study, we correlated pretreatment levels of IP-10 in liver biopsies (n = 73) and plasma (n = 265) with HCV RNA throughout therapy within a phase III treatment trial (DITTO-HCV). Low levels of plasma or intrahepatic IP-10 were strongly associated with a pronounced reduction of HCV RNA during the first 24 hours of treatment in all patients (P < 0.0001 and P = 0.002, respectively) as well as when patients were grouped as genotype 1 or 4 (P = 0.0008 and P = 0.01) and 2 or 3 (P = 0.002, and P = 0.02). Low plasma levels of IP-10 also were predictive of the absolute reduction of HCV RNA (P < 0.0001) and the maximum reduction of HCV RNA in the first 4 days of treatment (P < 0.0001) as well as sustained virological response (genotype 1/4; P < 0.0001). To corroborate the relationship between early viral decline and IP-10, pretreatment plasma samples from an independent phase IV trial for HCV genotypes 2/3 (NORDynamIC trial; n = 382) were analyzed. The results confirmed an association between IP-10 and the immediate reduction of HCV RNA in response to therapy (P = 0.006). In contrast, pretreatment levels of IP-10 in liver or in plasma did not affect the decline of HCV RNA between days 8 and 29, i.e., the second-phase decline, or later time points in any of these cohorts. CONCLUSION: In patients with chronic hepatitis C, low levels of intrahepatic and systemic IP-10 predict a favorable first-phase decline of HCV RNA during therapy with pegylated interferon and ribavirin for genotypes of HCV.

Inhibition of basal p38 or JNK activity enhances epithelial barrier function through differential modulation of claudin expression.

Tight junctions (TJs) form a barrier to the paracellular diffusion of ions and solutes across epithelia. Although transmembrane proteins of the claudin family have emerged as critical determinants of TJ permeability, little is known about the signaling pathways that control their expression. The aim of this study was to assess the role of three mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH(2)-terminal kinases (JNKs), and p38 kinases, in the regulation of epithelial barrier function and claudin expression in mammary epithelial cells. Addition of either PD169316 (a p38 inhibitor) or SP600125 (a JNK inhibitor) induced formation of domes (a phenomenon dependent on TJ barrier function) and enhanced transepithelial electrical resistance, whereas U0126 (an inhibitor of the ERK1/2 activators MEK1/MEK2) had no significant effect. Similar results were obtained using mechanistically unrelated p38 or JNK inhibitors. PD169316 increased the expression of claudin-4 and -8, whereas SP600125 increased claudin-4 and -9 and downregulated claudin-8. Silencing of p38alpha by isoform-specific small interfering RNAs increased claudin-4 and -8 mRNAs, whereas silencing of p38beta only increased claudin-4 mRNA. Silencing of either JNK1 or JNK2 increased claudin-9 mRNA expression while decreasing claudin-8 mRNA. Moreover, selective silencing of JNK2 increased claudin-4 and -7 mRNAs. Finally, both PD169316 and SP600125 inhibited the paracellular diffusion of Na(+) and Cl(-) across epithelial monolayers. Collectively, these results provide evidence that inhibition of either p38 or JNK enhances epithelial barrier function by selectively modulating claudin expression, implying that the basal activity of these MAPKs exerts a tonic effect on TJ ionic permeability.

Gene expression profile of Huh-7 cells expressing hepatitis C virus genotype 1b or 3a core proteins.

BACKGROUND: The liver disease expression in chronic hepatitis C patients is variable and may partially depend on the sequence of the infecting viral genotype. AIM: To identify some hepatitis C virus (HCV) genotype-specific virus-host interactions potentially leading to clinically significant consequences. METHODS: We compared the gene expression profile of Huh-7 cells transiently expressing the core protein of HCV genotype 1b and 3a using microarray technology. RESULTS: Thirty-two genes were overexpressed in Huh-7 transfected with the HCV genotype 1b core protein and 57 genes in cells transfected with the genotype 3a core protein. On the other hand, we found 20 genes downregulated by core 1b and 31 genes by core 3a. These included genes involved in lipid transport and metabolism, cell cycle, immune response and insulin signalling. CONCLUSION: The expression of HCV core proteins of different genotypes leads to a specific gene expression profile. This may account for the variable disease expression associated with HCV infection.

Hepatitis delta virus inhibits alpha interferon signaling.

UNLABELLED: Hepatitis delta virus (HDV) can cause severe acute and chronic liver disease in patients infected with hepatitis B virus. Interferon-alpha (IFN-alpha) is the only treatment reported to be effective in chronic hepatitis delta, albeit in a minority of patients. The molecular mechanisms underlying resistance to therapy are unclear. IFN-alpha-induced activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling cascade is essential for the induction of an antiviral state. Interference of HDV with the JAK-STAT pathway could be responsible for the IFN-alpha resistance in chronic hepatitis delta patients. We analyzed IFN-alpha-induced signal transduction through the JAK-STAT pathway in human hepatoma cells transfected with the complete HDV genome. The expression of IFN-alpha-stimulated genes was investigated with reverse transcription real-time polymerase chain reaction (PCR). STATs and JAKs activations were examined by immunofluorescence and immunoblot. The IFN-alpha-stimulated genes coding for the antiviral proteins myxovirus resistance A, double-stranded RNA (dsRNA)-activated protein kinase and 2',5'-oligoadenylate synthetase were down-regulated in HDV-transfected hepatoma cells in response to IFN-alpha treatment. HDV severely impaired the phosphorylation of both STAT1 and STAT2, thus preventing their accumulation in the nucleus. Furthermore, HDV blocked the IFN-alpha-stimulated tyrosine phosphorylation of IFN receptor-associated JAK kinase Tyk2, without affecting either the tyrosine phosphorylation of Jak1 or the expression of type I IFN receptor subunits. CONCLUSIONS: IFN-alpha-induced intracellular signaling is impaired in HDV-transfected human hepatoma cells. HDV subverts the effect of IFN-alpha by blocking Tyk2 activation, thereby resulting in selective impairment of activation and translocation to the nucleus of STAT1 and STAT2. Interference of HDV with IFN-alpha signaling could represent an important mechanism of viral persistence and treatment resistance.

Virus-induced over-expression of protein phosphatase 2A inhibits insulin signalling in chronic hepatitis C.

BACKGROUND/AIMS: Hepatitis C virus (HCV) infection disturbs glucose and lipid metabolism contributing to the development of liver steatosis, insulin resistance and type 2 diabetes mellitus. On the other hand, insulin resistance and steatosis have been found to be associated with increased rates of fibrosis progression and lower rates of response to interferon therapy in chronic hepatitis C (CHC). The molecular mechanisms contributing to insulin resistance in CHC are not well understood. We have shown previously that protein phosphatase 2A (PP2A) is over-expressed in biopsies from patients with CHC. In this study, we tested if PP2A over-expression leads to insulin resistance. METHODS: We studied insulin signalling in cell lines that allow the regulated over-expression of HCV proteins and of the PP2A catalytic subunit (PP2Ac). Insulin signalling and PP2Ac expression were also studied in HCV transgenic mice and in liver biopsies from patients with CHC. RESULTS: Over-expression of PP2Ac in cells inhibited insulin signalling by dephosphorylation of PKB/Akt. PP2Ac over-expression and impaired insulin signalling were found in the liver of HCV transgenic mice and in liver biopsies of patients with CHC. CONCLUSIONS: HCV-induced over-expression of PP2A in the liver contributes to the pathogenesis of insulin resistance in patients with CHC.

Hepatitis C viral kinetics in plasma and peripheral blood mononuclear cells during pegylated interferon-alpha2a/ribavirin therapy.

BACKGROUND/AIMS: Analysis of hepatitis C virus (HCV) RNA kinetics in compartments other than plasma may help in understanding HCV replication and identifying clinically significant patterns of treatment response. METHODS: After 6 weeks of pegylated interferon-alpha2a/ribavirin therapy, 74 chronic hepatitis C patients were randomized to individualized or standard treatments for another 42 weeks. HCV RNA was quantified in peripheral blood mononuclear cells (PBMCs) by TaqMan-based real-time PCR and compared to plasma HCV RNA. RESULTS: HCV RNA declines in PBMCs and plasma were comparable during the initial 12 weeks of therapy (Spearman's rank correlation range over different time points, 0.73-0.97). However, a delay of HCV RNA decay in PBMCs, expected if kinetics in PBMCs only reflected kinetics in plasma, was rarely observed. For many patients, HCV RNA decline in PBMCs started as early as in plasma and for some of them the kinetics strongly differed in the two compartments, hinting at a compartment-specific HCV replication and treatment effect. Fast viral decay in PBMCs was associated with sustained virological response, but viral kinetics in PBMCs added only minor predictive information compared with kinetics in plasma. CONCLUSIONS: Future kinetics studies of HCV RNA during therapy with new antivirals should take into account their compartment-specific effect.

Microarray analyses and molecular profiling of steatosis induction in immortalized human hepatocytes.

Hepatic steatosis is an important risk factor for the development of inflammation, fibrosis and impaired liver regeneration. The factors regulating lipid accumulation and driving hepatic steatosis toward inflammation, fibrosis and impaired regeneration are largely unknown. The aim of this study was to identify major alterations in gene expression occurring in steatotic hepatocytes, and to analyze how these changes impact cellular processes associated with steatosis. Microarray gene chips and RT-PCR were performed to analyze changes in gene expression induced in fatty human immortalized hepatocytes after treatment with 50 muM oleic acid for 7 days. Lipid metabolism and triglyceride accumulation in these cells was examined by Oil-Red-O staining, thin-layer chromatography (TLC) and immunofluorescence. Caspase 3 activity, BrdU incorporation and trypan blue exclusion were used to study apoptosis, proliferation and cell viability. Finally, quantitative analysis of signalling induced by insulin was performed by Western blot. Characterization of steatosis in three hepatocyte-derived cell lines indicated that the immortalized human hepatocytes (IHH) line was the most appropriate cell line for this study. Gene expression analysis showed significant alterations in the transcription of two major classes of genes involved either in cholesterol and fatty acid biosynthesis, as well as lipid export, or in apoptosis and cell proliferation. Such changes were functionally relevant, since TLC indicated that synthesis and accumulation of triglycerides were increased in steatotic cells, while synthesis of cholesterol and fatty acids were decreased. Lipid accumulation in IHH was associated with an increased apoptosis and an inhibition of cell proliferation and viability. No detectable changes in genes associated with insulin resistance were observed in steatotic cells, but signalling induced by insulin was more efficient in steatotic IHH as compared to control cells. We conclude that IHH represent a new valuable model of steatosis, not associated with insulin resistance, to study at both the genetic and functional level factors involved in the process of lipid accumulation and steatosis-associated liver injury.

The hepatitis C virus core protein of genotypes 3a and 1b downregulates insulin receptor substrate 1 through genotype-specific mechanisms.

UNLABELLED: Both molecular and clinical evidence support a link between HCV infection and insulin resistance. We examined the in vitro interaction between the HCV core protein of genotypes 3a and 1b with the insulin-signaling pathway. We measured the expression levels of insulin receptor substrate 1 (IRS-1), IRS-2, and other factors involved in the insulin signal pathway in a human hepatoma cell line (Huh-7) transiently expressing the HCV core protein of genotypes 3a or 1b by molecular biology and biochemical techniques. The IRS-1 (but not IRS-2) protein level was significantly reduced in Huh-7 expressing the core protein of both genotypes 3a and 1b, as compared to cells transfected with the empty vector. However, while the core protein of genotype 3a promoted IRS-1 degradation through the downregulation of peroxisome proliferator-activated receptor gamma (PPARgamma) and by upregulating the suppressor of cytokine signal 7 (SOCS-7), the core protein of genotype 1b activated the mammalian target of rapamycin (mTOR). We confirmed these findings by using agonists for PPARgamma (rosiglitazone) or short interfering RNAs for SOCS-7. CONCLUSION: Despite the small sequence divergence of the HCV core proteins of genotypes 3a and 1b, the 2 proteins appear to interfere with the insulin signaling pathway using genotype-specific mechanisms.

Real-time multiplex PCR assay to quantify hepatitis C virus RNA in peripheral blood mononuclear cells.

Ultrasensitive methods to measure very low levels of hepatitis C virus (HCV) RNA in biological samples may have diagnostic and prognostic significance and be useful to evaluate the response to antiviral treatment. A sensitive assay to quantify HCV RNA in peripheral blood mononuclear cells (PBMCs) was developed and validated using the iCycler iQ Detection System (Bio-Rad) coupled with TaqMan chemistry. HCV was co-amplified with the endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex reaction. Calculated PCR amplification efficiencies for both target and control genes were used in a mathematical model for relative quantitation of HCV RNA. A linear relationship between input RNA and C(T) values over 6 log dilutions was observed for both HCV- and GAPDH-specific products (R(2) > or = 0.99). As few as 1.5 IU/reaction could be detected, with high accuracy (CV < or= 3.94%) and reproducibility (CV < or = 2.20%). Quantitation of HCV RNA levels ranging from 10(3) to 10(7) IU/ml as measured in 47 plasma samples was highly correlated with values obtained by the COBAS Amplicor HCV Monitor test, v2.0 (Roche) (R(2) = 0.977). In conclusion, this assay provides an excellent tool to determine accurately HCV kinetics in PBMCs during antiviral therapy and to assess the long-term significance of different patterns of response to treatment.

An in vitro model of hepatitis C virus genotype 3a-associated triglycerides accumulation.

BACKGROUND/AIMS: The hepatitis C virus (HCV) induces lipid accumulation in vitro and in vivo. Although clinical observations are consistent with a direct effect of HCV genotype 3a on lipid metabolism, experimental systems have focused on the expression of HCV proteins of genotype 1. To extend these observations, we established an in vitro model expressing the HCV core of different genotypes. METHODS: The HCV core protein from patients with severe (genotype 3a) or no (genotypes 1b, 2a, 3h, 4h and 5a) liver steatosis was expressed in Huh7 cells. Core protein expression (by immunohistochemistry and immunoblot) and triglycerides accumulation (by Oil Red O stain and enzymatic measurement) were evaluated 48h after transfection. RESULTS: Although triglyceride accumulation occurred with genotypes 1b, 3a and 3h, the genotype 3a core protein expression resulted in the highest level of accumulation (i.e. about 3-fold with respect to 1b, and 2-fold with respect to 3h). This effect was not related to core protein expression levels and was abolished by culturing cells in lipid-free medium. CONCLUSIONS: Consistent with observations in chronic hepatitis C patients, the in vitro expression of HCV genotype 3a core protein is the ideal candidate model for studying the mechanisms of HCV-associated steatosis.

Roles of the C. elegans cyclophilin-like protein MOG-6 in MEP-1 binding and germline fates.

The switch from spermatogenesis to oogenesis in the Caenorhabditis elegans hermaphrodite requires mog-6, which post-transcriptionally represses the fem-3 RNA. In this study, we show that mog-6 codes for a divergent nuclear cyclophilin, in that a conserved domain is not required for its function in the sperm-oocyte switch. MOG-6 binds to the nuclear zinc finger protein MEP-1 through two separate domains that are essential for the role of MOG-6 in the sperm-oocyte switch. We propose that MOG-6 has a function distinct from that of prevailing cyclophilins and that its binding to MEP-1 is essential for germline sex determination. Finally, we found that gld-3 mog-6 mutants develop germline tumors, suggesting that mog-6 might function in the decision between mitosis and meiosis.

The MEP-1 zinc-finger protein acts with MOG DEAH box proteins to control gene expression via the fem-3 3' untranslated region in Caenorhabditis elegans.

Cell fates in the Caenorhabditis elegans germline are regulated, at least in part, at the posttranscriptional level. For example, the switch from spermatogenesis to oogenesis in the hermaphrodite relies on posttranscriptional repression of the fem-3 mRNA via its 3' untranslated region (UTR). Previous studies identified three DEAH box proteins, MOG-1, MOG-4, and MOG-5, that are critical for the fem-3 3' UTR control. Here we describe MEP-1, a zinc-finger protein that binds specifically to each of these three MOG proteins and that is required for repression by the fem-3 3' UTR in vivo. To investigate its in vivo function, we generated a mep-1 deletion mutant. The mep-1 null phenotype suggests a broad role for MEP-1 in C. elegans development, as it is associated with early larval arrest. In addition, mep-1 mutants can be defective in gonadogenesis and oocyte production when derived from a heterozygous mother. We suggest that MEP-1 acts together with the MOG proteins to repress fem-3 mRNA and that it also functions in other pathways to control development more broadly.