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Recently, with the advent of sofosbuvir/velpatasvir therapy, sustained virological response (SVR) can now be achieved even in patients with decompensated cirrhosis (dLC). However, the prognosis after SVR does not always improve in dLC, and appropriate indicators enabling prediction of prognosis is desired.Serum IP-10/CXCL10 levels were measured in 47 patients (15 chronic hepatitis [CH], 17 compensated cirrhosis [cLC], and 15 dLC) receiving direct acting antiviral (DAA) therapy, and their changes during the therapy were examined.All the patients achieved SVR. In patients with CH, the average IP-10 level was 367, 102, and 68 pg/ml respectively at baseline, at the end of therapy and at 12 weeks after SVR (SVR12), and was decreased upon DAA therapy (P 0.001). In patients with cLC, IP-10 was respectively 215, 91, and 77 pg/ml, and was decreased upon DAA therapy (P 0.001) while it was 283, 131, and 182 pg/ml in patients with dLC and there was no evident decrease (P = 0.55). When patients with dLC were further classified depending on the difference in Child-Pugh (CP) score improvement at SVR12, a significant decrease in IP-10 was observed after treatment in those with improvement (P = 0.023) while a significant increase was observed in those without improvement (P = 0.016).While serum IP-10 level was decreased in patients with CH/cLC and dLC with post-SVR-CP improvement following SVR, it was increased in patients with dLC without post-SVR CP improvement. The result indicates that IP-10 dynamics may be useful for predicting liver function after DAA therapy.

AIM Recently, serum hepatitis B virus (HBV)-RNA has been reported to be detectable even when HBV particle production is inhibited by nucleot(s)ide analogues (NAs). However, the dynamics of the HBV-RNA sequence compared with those of HBV-DNA during the emergence of antiviral resistance are yet to be elucidated. METHODS First, we quantified serum HBV-RNA in 181 infected patients, and its relationships with clinical characteristics as well as HBV markers were investigated. Next, we undertook simultaneous deep sequencing of HBV-RNA/HBV-DNA and their dynamics among four patients receiving NA therapy who were experiencing viral breakthrough. RESULTS Serum HBV-RNA was detected in 25% (31/123) of cases among patients with HBV without NAs, and the detection rate was significantly high in hepatitis B e antigen-positive cases with high viral activity. In patients with chronic hepatitis, hepatitis B core-related antigen was significantly correlated with serum HBV-RNA irrespective of NA use. In the analysis of the four patients experiencing viral breakthrough, no NA resistance mutation was detected in the serum HBV-RNA immediately before the breakthrough. However, NA-resistant sequences appeared at the rates of 0%, 3%, 14%, and 100%, and the NA-resistant HBV-RNA sequence rate was correlated with the peak HBV-DNA titer multiplied by the HBV-DNA detection duration during the breakthrough (R2 = 0.978) observed before redisappearance of HBV-DNA following the addition of new NA. CONCLUSION Serum HBV-RNA could reflect the transcriptional activity of covalently closed circular DNA and hepatitis B core-related antigen. The dynamics of HBV-RNA could help understanding of the turnover process of HBV covalently closed circular DNA in the liver.

Aim Liver fibrosis is a risk factor for hepatocellular carcinoma (HCC), but at what fibrotic stage the risk for HCC is increased has been poorly investigated quantitatively. This study aimed to determine the appropriate cut-off value of liver stiffness for HCC concurrence by FibroScan, and its clinical significance in hepatitis B virus (HBV), hepatitis C virus (HCV) and non-B, non-C (NBNC) liver disease. Methods Subjects comprised 1002 cases (246 with HCC and 756 without HCC) with chronic liver disease (HBV, 104; HCV, 722; and NBNC, 176). Results Liver stiffness was significantly greater in all groups with HCC, and the determined cut-off value for HCC concurrence was more than 12.0 kPa in those with HCV, more than 8.5 kPa in those with HBV and more than 12.0 kPa in those with NBNC. Liver stiffness of more than 12.0 kPa was an independent risk factor for new HCC development in HCV. For HCV, risk factors for HCC concurrence were old age, male sex, low albumin, low platelets and liver stiffness, while for HBV they were old age, low platelets and liver stiffness, and for NBNC they were old age, elevated α-fetoprotein and liver stiffness. Conclusion Liver stiffness cut-off values and their association with HCC concurrence were different depending on the etiology. In HCV, liver stiffness of more than 12.0 kPa was an independent risk factor for new HCC development. Collectively, determining the fibrotic cut-off values for HCC concurrence would be important in evaluating HCC risks.

Aim To investigate whether the patients with hypovascular liver nodules determined on the arterial phase and hypointensity on the hepatocyte phase gadoxetic acid-enhanced magnetic resonance imaging (hypovascular hypointense nodules) are at increased risk of hepatocarcinogenesis, we assessed subsequent typical hepatocellular carcinoma (HCC) development at any sites of the liver with and without such nodules. Methods One hundred and twenty-seven patients with chronic hepatitis B or C and without a history of HCC, including 68 with liver cirrhosis, were divided into those with (non-clean liver group, n = 18) and without (clean liver group, n = 109) hypovascular hypointense nodules. All the patients were followed up for 3 years, and HCC development rates and risk factors were analyzed with the Kaplan–Meier method and the Cox proportional hazard model, respectively. Results A total of 17 patients (10 in the non-clean liver group and seven in the clean liver group) developed typical HCC. Cumulative 3-year rates of HCC development were 55.5% in the non-clean liver group and 6.4% in the clean liver group (P

Aim Daclatasvir, a non-structural (NS)5A replication complex inhibitor, is a potent and promising direct antiviral agent (DAA) for hepatitis C virus (HCV), being most effective in genotype 1b infection. Although it is known that genotype 1b viruses with Y93H and/or L31M/V/F mutations have strong resistance to daclatasvir, it is not known whether there are some clinical background conditions that favor the occurrence of HCV carrying those NS5A mutations. Methods In this study, we carried out deep sequencing analysis of stored sera to determine the presence and significance of daclatasvir-resistant mutants in 110 genotype 1b HCV-infected patients with no previous daclatasvir treatment. Results Deep sequencing analysis revealed that the NS5A L31M/V/F and Y93H mutations were present in 13 (11.8%) and 34 (30.9%) of the 110 patients, respectively, and significantly more frequently than in the control plasmid. Simultaneous L31M/V/F and Y93H mutations were detected in four of the 110 patients (3.6%). When the clinical relevance of NS5A resistance was investigated, Y93H was significantly correlated with the IL28B major (TT) genotype of the host (P = 0.042). Conclusion Y93H was detected frequently by deep sequencing in daclatasvir treatment-naive patients. Importantly, it seems that the IL28B status of the patients may influence the presence of Y93H mutations, resulting in different treatment responses to daclatasvir.

Aim Prediction of treatment responses to pegylated interferon (PEG IFN) plus ribavirin (RBV) therapy is uncertain for genotype 1b chronic hepatitis C. Methods In this study, 96 patients were investigated for the correlation between 36 pretreatment serum chemokine/cytokine levels and PEG IFN/RBV treatment efficacy by a sandwich enzyme-linked immunoassay (ELISA) and a bead array. Results First, chemokines/cytokines were measured semiquantitatively by sandwich ELISA in 31 randomly-selected patients and the serum regulated on activation normal T-cell expressed and secreted (RANTES) level was found to be significantly higher in the sustained virological response (SVR) group than the non-SVR group (P = 0.048). Precise RANTES measurement in all 96 patients using a bead array confirmed this correlation (P = 0.002). However, the genetic RANTES haplotype was not significantly related to the serum level. The serum RANTES level was extracted by multivariate analysis (odds ratio = 4.09, 95% confidence interval = 1.02–16.5, P = 0.048) as an independent variable contributing to SVR. Conclusion The serum RANTES level is an important determinant influencing the virological response to PEG IFN/RBV therapy in chronic hepatitis C.

Aim: Traditional herbal medicines have been used for several thousand years in China and other Asian countries. In this study we screened herbal drugs and their purified compounds, using the Feo replicon system, to determine their effects on in vitro HCV replication. Methods: We screened herbal drugs and their purified extracts for the activities to suppress hepatitis C virus (HCV) replication using an HCV replicon system that expressed chimeric firefly luciferase reporter and neomycin phosphotransferase (Feo) genes. We tested extracts and 13 purified compounds from the following herbs: Glycyrrhizae radix; Rehmanniae radix; Paeoniae radix; Artemisiae capillari spica; and Rhei rhizoma. Results: The HCV replication was significantly and dose-dependently suppressed by two purified compounds, isoliquiritigenin and glycycoumarin, which were from Glycyrrhizae radix. Dose-effect analyses showed that 50% effective concentrations were 6.2 ± 1.0 µg/mL and 15.5 ± 0.8 µg/mL for isoliquiritigenin and glycycoumarin, respectively. The MTS assay did not show any effect on cell growth and viability at these effective concentrations, indicating that the effects of the two compounds were specific to HCV replication. These two compounds did not affect the HCV IRES-dependent translation nor did they show synergistic action with interferon-alpha. Conclusion: Two purified herbal extracts, isoliquiritigenin and glycycoumarin, specifically suppressed in vitro HCV replication. Further elucidation of their mechanisms of action and evaluation of in vivo effects and safety might constitute a new anti-HCV therapeutics.

Backgrounds Interferon (IFN)-alpha is represented by several structurally related subtypes that show different antiviral and anti-tumor effects. Here, we analyzed differential effects of IFN-alpha subtypes on intracellular hepatitis C virus (HCV) replication using HCV subgenomic replicon system as a model. Methods Huh7 and HeLa cells supporting expression of HCV replicon were treated with various concentrations of five recombinant human IFN-alpha subtypes 1, 2, 5, 8, and 10, and with IFN-alpha con1. The effects of IFNs on various cell-signaling pathways were assayed by using ISRE-, GAS-, AP1-, NF-kappa B-, CRE-, and SRE-luciferase reporter plasmids. Results Each IFN-alpha subtype suppressed HCV replication in a dose-dependent manner. Among them, IFN-alpha8 was the most effective, while IFN-alpha1 was the least effective with 50% inhibitory concentrations of 0.123 IU/ml versus 0.375 IU/ml, respectively. These differential effects against HCV replication did not correlate with levels of the IFN-responsive ISRE or GAS reporter activities, nor they did activate the other reporters, AP1, NF-kappa B, CRE and SRE. Conclusion There were divergent effects of IFN-alpha subtypes against HCV replication that may be through JAK-STAT-independent pathways. Exploring further mechanisms of action may elucidate IFN-mediated cellular antiviral mechanisms.

Primary biliary cirrhosis (PBC) is one of the most important autoimmune liver diseases but the etiology and pathogenesis remain unknown. In this study, we analyzed differential mRNA expression in the liver of a patient with PBC using suppression subtractive hybridization to identify overexpressed genes. Overexpression of mRNA transcripts from mitochondrial DNA was observed in the PBC liver, compared to normal liver. To explore the mechanism of increased mitochondrial transcription, we investigated the mRNA levels of nuclear DNA-encoded regulator molecules of mitochondrial gene expression in 60 liver biopsy samples from various diseases, including PBC, using competitive RT-PCR. Increased expression of mitochondrial transcriptional factor A (mtTFA) and mitochondrial nuclear respiratory factor 1 (NRF-1) mRNA was demonstrated in PBC liver compared to other liver diseases, while NRF-1 coactivator 1, PGC-1 was suppressed. Mitochondrial DNA-encoded mRNA molecules are overexpressed in the PBC liver, and this is associated with up-regulation of mitochondrial transcription factor mtTFA and its transactivator NRF-1. Further studies are needed to focus on the relevance of this perturbation of mitochondrial gene expression in the pathogenesis of PBC.

Aim: Hepatitis C virus (HCV), which infects an estimated 170 million people worldwide, is a major cause of chronic liver disease. The current standard therapy for chronic hepatitis C is based on pegylated interferon (IFN)α in combination with ribavirin. However, the success rate remains at approximately 50%. Therefore, alternative agents are needed for the treatment of HCV infection. Methods: Using an HCV-1b subgenomic replicon cell culture system (Huh7/Rep-Feo), we found that griseofulvin, an oral antifungal agent, suppressed HCV-RNA replication and protein expression in a dose-dependent manner. We also found that griseofulvin suppressed the replication of infectious HCV JFH-1. A combination of IFNα and griseofulvin exhibited a synergistic inhibitory effect in Huh7/Rep-Feo cells. Results: We found that griseofulvin blocked the cell cycle at the G2/M phase in the HCV subgenomic replicon cells, but did not inhibit HCV internal ribosome entry site-dependent translation. Conclusion: Our results suggest that griseofulvin may represent a new approach to the development of a novel therapy for HCV infection.