Sensitive Assay for Quantification of Hepatitis B Virus Mutants by Use of a Minor Groove Binder Probe and Peptide Nucleic Acids

Lamivudine is the first nucleoside analogue that was shown to have a potent effect on hepatitis B virus (HBV). However, the emergence of resistant or cross-resistant mutants to nucleos(t)ide analogues remains a serious problem. Several assays for the detection and quantification of antiviral resistant mutants have been reported, but it has been difficult to measure the amounts of mutants accurately, especially when the target strain is minor in the mixed population. It has been shown that accurate measurement of a minor strain is difficult as long as matching reaction with a single probe was included in the assay. We developed a new method for the quantification of lamivudine-resistant strains in a mixed virus population by real-time PCR using minor groove binder probes and peptide nucleic acids, and we achieved a wide and measurable range from 3 to 10 log10 copies/ml and a high sensitivity with a discriminative limit of 0.01% of the predominant strain. The clinical significance of measuring substitutions of not only M204 but also L180 residues of HBV polymerase was demonstrated by this method. This assay increases the versatility of a sensitive method for the quantification of a single nucleotide mutation in a heterogeneous population

Constitutive aryl hydrocarbon receptor signaling constrains type I interferon–mediated antiviral innate defense

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic activity of many environmental xenobiotics. However, its role in innate immune responses during viral infection is not fully understood. Here we demonstrate that constitutive AHR signaling negatively regulates the type I interferon (IFN-I) response during infection with various types of virus. Virus-induced IFN-β production was enhanced in AHR-deficient cells and mice and resulted in restricted viral replication. We found that AHR upregulates expression of the ADP-ribosylase TIPARP, which in turn causes downregulation of the IFN-I response. Mechanistically, TIPARP interacted with the kinase TBK1 and suppressed its activity by ADP-ribosylation. Thus, this study reveals the physiological importance of endogenous activation of AHR signaling in shaping the IFN-I-mediated innate response and, further, suggests that the AHR-TIPARP axis is a potential therapeutic target for enhancing antiviral responses.Supplementary materials are available on the publisher's website

The influence of hepatitis B DNA level and antiviral therapy on recurrence after initial curative treatment in patients with hepatocellular carcinoma

Background. Prediction and prevention of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) recurrence is an important clinical issue. We investigated whether HBV DNA level and antiviral therapy are associated with HCC recurrence. Methods. This retrospective study involved 103 patients who underwent hepatic resection or radiofrequency ablation for initial HCC. Patients were divided into four groups. Thirty had high serum HBV DNA levels (>4 log10 copies/mL) and had not received antiviral therapy (high virus group; HVG). Thirty-four had low HBV DNA levels (≤4 log10 copies/mL) and had not received antiviral therapy (low virus group; LVG). Twenty received antiviral therapy after HCC developed (therapeutic group A, TG-A). Nineteen received antiviral therapy before HCC developed (therapeutic group B, TG-B). Results. Cumulative HCC recurrence rates at 3 years in the HVG, LVG, TG-B, and TG-A were 71.1%, 42.2%, 42.3%, and 52.0%, respectively. Recurrence rates differed significantly between the HVG and LVG (P = 0.016) and between the HVG and TG-B (P = 0.008). Recurrence rate in the TG-A was marginally lower than in the HVG (P = 0.10). On multivariate analysis, high serum hepatitis B virus DNA levels (hazard ratio: HR, 2.67; 95% CI, 1.31-5.47; P = 0.007) and absence of antiviral therapy (HR, 2.57; 95% CI, 1.34-4.94; P = 0.005) were independent risk factors for hepatocellular carcinoma recurrence. Conclusion. HBV DNA level and antiviral therapy are associated with HCC recurrence. For patients with high HBV DNA levels, antiviral therapy before the development of HCC is important for prevention of recurrence

A Phase I Study of Combination Therapy with Sorafenib and 5-Fluorouracil in Patients with Advanced Hepatocellular Carcinoma

Background and aims: Sorafenib is the first molecular targeted drug approved for the treatment of advanced hepatocellular carcinoma (HCC) and is a potent small molecule inhibitor of multiple kinases. Combination therapy with sorafenib and other cytotoxic agents for HCC may result in additive anticancer activity. The purpose of this phase I study was to investigate the safety and tolerability of combination therapy with sorafenib and 5-fluorouracil (5-FU) and to determine the optimum dose of 5-FU for a phase II trial. Methods: This phase I study used a conventional 3 + 3 dose-escalation design. The primary endpoint was to determine the maximum tolerated dose (MTD) of 5-FU in combination with sorafenib and to determine the recommended dosage (RD) for phase II. The secondary endpoints evaluated were toxicity and the tumor response rate. All patients received 800 mg of sorafenib daily and three different dosages of 5-FU (250, 350, and 450 mg/m2/day) for 20 days by intravenous infusion in 1 month as one cycle. Results: Twelve patients with advanced HCC were evaluated. The MTD of 5-FU in combination with sorafenib was 450 mg/m2/day, and 350 mg/m2/day was selected as the RD for a phase II study. Thrombocytopenia, stomatitis, and hand-foot skin reaction were observed as grade 3 adverse events. Nine patients achieved stable disease (75%), and three patients (25%) were judged to have progressive disease. The disease control rate was 75%. Conclusions: Combination therapy with sorafenib and 5-FU appears to be well tolerated and may have the potential to be an option for advanced HCC

Efficacy and safety of daclatasvir and asunaprevir combination therapy in chronic hemodialysis patients with chronic hepatitis C

Background: HCV infection in chronic hemodialysis patients is high, has a poor prognosis and high risk of renal graft failure, and requires nosocomial infection control measures. However, options of anti-HCV therapy in such patients are limited and unsatisfactory. In this study, we report effectiveness and safety of HCV-NS5A-inhibitor daclatasvir (DCV) and protease-inhibitor asunaprevir (ASV) combination therapy for hemodialysis patients with HCV infection. Methods: This study was registered at the UMIN Clinical Trials Registry as UMIN000016355. Thirty-four dialysis patients were treated with DCV/ASV combination therapy between January 2015 and November 2015. Of those, 21 patients who were followed more than 12 weeks after treatment ended were included. We evaluated the 12-week sustained virologic response (SVR12) and adverse events during treatment. Results: Of the 21 patients, four had compensated liver cirrhosis and three had resistance-associated variant of NS5A (NS5A RAVs)-Y93H at baseline. Overall, total of 95.5 % (20/21) of the patients achieved SVR12. Of note, all patients with cirrhosis or NS5A RAVs achieved SVR12. One relapser patient at 4 weeks post-treatment had NS3 D168E RAVs at baseline. A total of 20 patients (95.5 %) completed the 24-week therapy. One patient discontinued treatment at week 12 due to ALT elevations and achieved SVR12. Conclusions: DAV and ASV combination therapy for chronic hemodialysis patients with HCV infection was highly effective and well tolerated, even in elderly patients and patients with liver cirrhosis and NS5A-RAVs