Genotype-specific mutations in the polymerase gene of hepatitis B virus potentially associated with resistance to oral antiviral therapy.

The evolution of hepatitis B virus (HBV) and the role of different variants during antiviral therapy may be influenced by HBV genotype. We have therefore analysed substitutions potentially related to nucleos(t)ide analogues (NAs) resistance at 42 positions within RT-region in a cohort of patients with chronic hepatitis B in relation to HBV-genotype. RT mutations analysis was performed by direct sequencing in 200 NAs-naïve patients and in 64 LAM or LAM+ADV experienced patients with NAs resistance, infected mainly by HBV-genotypes D and A. 27 polymorphic-sites were identified among the 42 positions analysed and 64 novel mutations were detected in 23 positions. Genotype-D displayed the highest mutation frequency (6.4%) among all HBV-genotypes analysed. Single or multiple mutations were detected in 80% of naïve patients. Overall, the most frequent single mutations were at residues rt54, rt53 and rt91 which may associate with significantly lower HBV-DNA levels (p=0.001). Comparison with sequencing data of patients failing LMV or LAM+ADV therapy revealed an higher frequency of novel genotype-specific mutations if compared with naïve patients: 3 mutations under LAM monotherapy in HBV-D (rtS85F; rtL91I; rtC256G) and 3 mutations under ADV therapy in HBV-A (rtI53V; rtW153R; rtF221Y). In HBV-D treated patients the dominant resistance mutation was rtL80V (31.4%) and rtM204I (60%) in LAM+ADV group while LAM-treated patients showed a preference of rtM204V (51.9%). Interestingly, none of HBV-A patients had mutation rtM204I under ADV add-on treatment but all of them had the "V" AA substitution. These results suggested that in patients with CHB, HBV-genotype might be relevant in the evolution and development of drug resistance showing also different mutation patterns in the YMDD motif between HBV genotype D and A.

Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury.

BACKGROUND: Hepatocytes and stem cells transplantation may be an alternative to liver transplantation in acute or chronic liver disease. We aimed to evaluate the therapeutic potential of mesenchymal stem cells from human umbilical cord (UCMSCs), a readily available source of mesenchymal stem cells, in the CCl4-induced acute liver injury model. METHODS: Mesenchymal stem cells profile was analyzed by flow cytometry. In order to evaluate the capability of our UCMSCs to differentiate in hepatocytes, cells were seeded on three different supports, untreated plastic support, MatrigelTM and human liver acellular matrix. Cells were analyzed by immunocitochemistry for alpha-fetoprotein and albumin expression, qPCR for hepatocyte markers gene expression, Periodic Acid-Schiff staining for glycogen storage, ELISA for albumin detection and colorimetric assay for urea secretion.To assess the effects of undifferentiated UCMSCs in hepatic regeneration after an acute liver injury, we transplanted them via tail vein in mice injected intraperitoneally with a single dose of CCl4. Livers were analyzed by histological evaluation for damage quantification, immunostaining for Kupffer and stellate cells/liver myofibroblasts activation and for UCMSCs homing. Pro- and anti-inflammatory cytokines gene expression was evaluated by qPCR analysis and antioxidant enzyme activity was measured by catalase quantification.Data were analyzed by Mann-Whitney U-test, Kruskal-Wallis test and Cuzick's test followed by Bonferroni correction for multiple comparisons. RESULTS: We have standardized the isolation procedure to obtain a cell population with hepatogenic properties prior to in vivo transplantation. When subjected to hepatogenic differentiation on untreated plastic support, UCMSCs differentiated in hepatocyte-like cells as demonstrated by their morphology, progressive up-regulation of mature hepatocyte markers, glycogen storage, albumin and urea secretion. However, cells seeded on 3D-supports showed a minor or negligible differentiation capacity.UCMSCs-transplanted mice showed a more rapid damage resolution, as shown by histological analysis, with a lower inflammation level and an increased catalase activity compared to CCl4-treated mice. CONCLUSIONS: Our findings show that UCMSCs can be reliably isolated, have hepatogenic properties and following systemic administration are able to accelerate the resolution of an acute liver injury without any differentiation and manipulation. These features make UCMSCs strong candidates for future application in regenerative medicine for human acute liver disease.

Reciprocal interference between insulin and interferon-alpha signaling in hepatic cells: a vicious circle of clinical significance?

UNLABELLED: Insulin resistance (IR) is common in chronic hepatitis C (CHC) and associates with reduced virological response to pegylated-interferon (PEG-IFN)/ribavirin therapy, but the underlying mechanisms are still unclear. We have previously shown that, in CHC patients, insulin plasma levels are inversely related to antiviral effect induced by PEG-IFN. Therefore, we investigated the in vitro effect of insulin on interferon alpha (IFN-α) intracellular signaling as well as that of IFN-α on insulin signaling. HepG2 cells, preincubated with or without insulin, were stimulated with IFN-α2b and messenger RNA (mRNA) and protein expression of IFN-stimulated genes (ISGs) were measured at different timepoints. The role of intracellular suppressors of cytokine signaling 3 (SOCS3) was evaluated with the small interfering RNA (siRNA) strategy. To assess the effect of IFN-α on insulin signaling, HepG2 were preincubated with or without IFN before addition of insulin and cells were then analyzed for IRS-1 and for Akt/PKB Ser473 phosphorylation. Insulin (100 and 1000 nM) significantly reduced in a dose-dependent fashion IFN-induced gene expression of PKR (P=0.017 and P=0.0017, respectively), MxA (P=0.0103 and P=0.00186), and 2'-5' oligoadenylatesynthetase 1 (OAS-1) (P=0.002 and P=0.006). Insulin also reduced IFN-α-induced PKR protein expression. Although insulin was confirmed to increase SOCS3 expression, siRNA SOCS3 did not restore ISG expression after insulin treatment. IFN-α was found to reduce, in a dose-dependent fashion, IRS-1 gene expression as well as Akt/PKB Ser473 phosphorylation induced by insulin. CONCLUSION: These results provide evidence of reciprocal interference between insulin and IFN-α signaling in liver cells. These findings may contribute to understand the role of insulin in CHC: IR might be favored by endogenous cytokines including IFN-α, and the resulting hyperinsulinemia then reduces the antiviral response to exogenous IFN in a vicious circle of clinical significance.

Comparable performance of TMA and Real-Time PCR in detecting minimal residual hepatitis C viraemia at the end of antiviral therapy.

BACKGROUND: Antiviral therapy for chronic hepatitis C may cause transient on-treatment response followed by post-treatment relapse. OBJECTIVES: We have compared the prognostic value for post-treatment relapse of minimal hepatitis C residual viraemia detected at end-of-therapy by transcription mediated assay (TMA) and by Abbott RealTime PCR HCV assay. STUDY DESIGN: Minimal residual viraemia was investigated in 202 HCV patients who had completed a full course of Pegylated Interferon (PEG-IFN) plus ribavirin and were HCV-RNA negative by conventional PCR in two separate serum samples obtained during the last week of therapy and the results were then correlated with post-treatment outcome. RESULTS: Minimal residual viraemia was detected in 22/202 (11%, 95% CI: 7-16%) and in 28/202 (13.8%, 95% CI 10-19%) patients by TMA and by Abbott RealTime HCV assay, respectively, with 92% concordant results. Post-treatment relapse was seen in 81.8% (95% CI: 60-93%) of TMA positive and in 82.1% (95% CI: 64-92%) of Abbott RealTime HCV assay positive cases compared to 16.6% (95% CI: 12-23%) of TMA negative and 17.2% (95% CI: 12-23%) of Abbott RealTime HCV assay negative patients. CONCLUSIONS: These results indicate that TMA and the Abbott RealTime HCV assay have comparable sensitivity and specificity in identifying minimal residual viraemia at the end of antiviral therapy, with excellent predictive value for post-treatment relapse.

Hepatic steatosis in hepatitis C is a storage disease due to HCV interaction with microsomal triglyceride transfer protein (MTP).

Liver steatosis is a frequent histological feature in patients chronically infected with hepatitis C virus (HCV). The relationship between HCV and hepatic steatosis seems to be the result of both epigenetic and genetic factors. In vivo and in vitro studies have shown that HCV can alter intrahepatic lipid metabolism by affecting lipid synthesis, oxidative stress, lipid peroxidation, insulin resistance and the assembly and secretion of VLDL. Many studies suggest that HCV-related steatosis might be the result of a direct interaction between the virus and MTP. It has been demonstrated that MTP is critical for the secretion of HCV particles and that inhibition of its lipid transfer activity reduces HCV production. However, higher degrees of hepatic steatosis were found in chronic hepatitis C patients carrying the T allele of MTP -493G/T polymorphism that seems to be associated with increased MTP transcription. We propose here that liver steatosis in hepatitis C could be a storage disease induced by the effects of the virus and of its proteins on the intracellular lipid machinery and on MTP. Available data support the hypothesis that HCV may modulate MTP expression and activity through a number of mechanisms such as inhibition of its activity and transcriptional control. Initial up regulation could favour propagation of HCV while down regulation in chronic phase could cause impairment of triglyceride secretion and excessive lipid accumulation, with abnormal lipid droplets facilitating the "storage" of virus particles for persistent infection.

Microsomal triglyceride transfer protein polymorphism (-493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C.

BACKGROUND: Hepatic steatosis may promote progression of chronic hepatitis C (CHC). Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of ApoB lipoprotein and is implicated in hepatitis C virus (HCV)-related steatosis. The MTP -493G/T polymorphism may promote liver fat accumulation, but its role in HCV-related steatosis is still unclear. METHODS: Two hundred ninety-eight CHC patients were studied and genotyped for MTP -493G/T variants. Hepatic MTP mRNA expression and activity were determined in a subgroup. RESULTS: Patients with grades 2/3 steatosis were older, had a higher body mass index (BMI), more advanced fibrosis and lower MTP mRNA expression and carried more often HCV genotype 3 and the MTP T allele. Age, BMI, HCV-3 and MTP T allele [odds ratio (OR) 2.05; 95% confidence interval (CI) 1.2-3.53; P=0.009] were independent risk factors for steatosis grades 2/3, and in HCV genotype non-3 patients, the MTP T allele was the strongest predictor for steatosis grade 2/3 (OR 2.17; 95% CI 1.22-3.86; P=0.008). Moreover, TT carriers had higher high-density lipoprotein (65.6+/-14.6 vs 56.1+/-16.2 mg/dl; P=0.003) and apolipoprotein AI (1.80+/-0.3 vs 1.60+/-0.3 g/L; P=0.005) levels than G allele carriers. CONCLUSIONS: Chronic hepatitis C patients with the MTP -493T allele reveal higher grades of steatosis, indicating a relevant contribution to liver fat accumulation, particularly in HCV non-3 patients.

Liver microsomal triglyceride transfer protein is involved in hepatitis C liver steatosis.

BACKGROUND & AIMS: Hepatic steatosis is frequent in chronic hepatitis C. Several mechanisms might be implicated, including metabolic cofactors and direct viral effects on intracellular lipid pathways. In a transgenic mouse model, hepatitis C virus (HCV) was shown to inhibit microsomal triglyceride transfer protein (MTP) activity, which is essential for hepatic lipoprotein assembly and secretion. No data are available on liver MTP activity in HCV-infected patients. We therefore investigated liver MTP gene expression and its lipid transfer activity in untreated cases infected with the major HCV genotypes showing variable degrees of hepatic steatosis. METHODS: MTP messenger RNA (mRNA) levels were measured by real-time polymerase chain reaction, and MTP activity was assessed by fluorescent assay in liver biopsy specimens of 58 HCV-positive patients. A set of metabolic and serum lipid markers was also measured at the time of liver biopsies. RESULTS: MTP mRNA levels showed a statistically significant (P = .001) inverse correlation with the degree of steatosis, independently of the HCV genotype. MTP mRNA levels also had an inverse correlation with serum insulin (P = .0002), homeostasis model assessment-insulin resistance (HOMA-IR) (P = .005), and body mass index (P = .02) in patients with HCV-1 and HCV-2 and with serum HCV-RNA (P = .02) in HCV-3 patients. Liver MTP-specific activity was significantly reduced in HCV-3 patients compared with those with other HCV genotypes (P = .004) and correlated with reduced serum cholesterol, apo B, and low-density lipoproteins. CONCLUSIONS: MTP may play a central role in HCV-related steatosis, being modulated by different genotype-specific mechanisms, mainly hyperinsulinemia in non-HCV-3 patients, and more profound and direct virus-related effects in HCV-3-infected individuals.