Yuhki Yamaguchi

Hepatitis C virus core protein inhibits deoxycholic acid-mediated apoptosis despite generating mitochondrial reactive oxygen species

BackgroundHepatitis C virus (HCV) core protein is known to cause oxidative stress and alter apoptosis pathways. However, the apoptosis results are inconsistent, and the real significance of oxidative stress is not well known. The aim of this study was twofold. First, we wanted to confirm whether core-induced oxidative stress was really significant enough to cause DNA damage, and whether it induced cellular antioxidant responses. Second, we wanted to evaluate whether this core-induced oxidative stress and the antioxidant response to it was responsible for apoptosis changes.MethodsHCV core protein was expressed under control of the Tet-Off promoter in Huh-7 cells and HeLa cells. We chose to use deoxycholic acid (DCA) as a model because it is known to produce both reactive oxygen species (ROS) and apoptosis.ResultsCore expression uniformly increased ROS and 8-hydroxy-2′-deoxyguanosine (8-OHdG) under basal and DCA-stimulated conditions. Core protein expression also increased manganese superoxide dismutase levels. Core protein inhibited DCA-mediated mitochondrial membrane depolarization and DCA-mediated activation of caspase-9 and caspase-3, despite the increase in ROS by DCA. Core protein inhibited DCA-mediated apoptosis by increasing Bcl-xL protein and decreasing Bax protein, without affecting the proportion of Bax between mitochondria and cytosol, resulting in suppression of cytochrome c release from mitochondria into cytoplasm.ConclusionsHCV core protein induces oxidative DNA damage, whereas it inhibits apoptosis that is accompanied by enhancement of ROS production. Thus, oxidative stress and apoptosis modulation by core protein are independent of each other.

In situ detection of oxidized n-3 polyunsaturated fatty acids in chronic hepatitis C: correlation with hepatic steatosis

BackgroundOxidative stress contributes to the pathogenesis of chronic hepatitis C. The aim of this study was to assess the peroxidation of n-3 polyunsaturated fatty acids (PUFAs) in the liver and its relation to hepatic steatosis in chronic hepatitis C.MethodsWe immunohistochemically detected malondialdehyde (MDA)-, 4-hydroxy-2-nonenal (HNE)-, and 4-hydroxy-2-hexenal (HHE)-protein adducts in liver biopsy specimens from 55 patients with chronic hepatitis C. Cells stained positively for HHE-protein adducts were quantified using computer-based image analysis. Fatty-acid composition was determined, by gas chromatography, for the noncancerous portions of resected livers, with or without steatosis, obtained from two patients with hepatitis C virus-associated hepatocellular carcinoma.ResultsThe detection rate of HHE-protein adducts (63.6%) was significantly higher than that of MDA-protein adducts (21.8%; P < 0.001) or HNE-protein adducts (29.1%; P < 0.001). Areas positively stained for HHE-protein adducts (HHE-positive areas) were significantly larger in 18 patients with steatosis (6.2 ± 3.6%) than in 17 patients without steatosis (3.4 ± 2.6%; P = 0.01). Resected liver tissue with steatosis showed a larger HHE-positive area (18.6%) and higher ratio of n-6 PUFA content to n-3 PUFA content (3 : 1) than liver tissue without steatosis (7.2%; 2 : 3). On multivariate analysis, the HHE-positive area (odds ratio, 1.55; 95% confidence interval [CI], 1.08–2.23; P = 0.019) was a factor associated with the presence of hepatic steatosis.ConclusionsHHE-protein adducts, which are a good marker for oxidative stress, are associated with steatosis in chronic hepatitis C.

Expression of type I interferon receptor in liver and peripheral blood mononuclear cells in chronic hepatitis C patients.

To assess whether peripheral blood mononuclear cells (PBMCs) can be substituted for liver tissue in monitoring the hepatic expression of interferon receptors (IFNAR1 and/or IFNAR2) in chronic hepatitis C, we quantified the mRNA of both subunits, using the competitive polymerase chain reaction, in the liver specimens from 37 chronic hepatitis C patients and in PBMCs from 29 of 37 patients. PBMCs from 10 healthy subjects were also quantified for both subunits. Hepatic expression of IFNAR1 and IFNAR2 were more frequent than PBMCs: IFNAR1, 23/27 (85%) vs 7/19 (37%), P = 0.0021, IFNAR2, 34/37 (92%) vs 19/29 (66%), P = 0.0182. Neither subunits was detected in PBMCs from control subjects. The expression level of IFNAR2 in the liver was related to that in PBMCs (r = 0.613, P = 0.0052). These results suggest that hepatitis C virus infection may up-regulate the expression of the type I interferon receptor and that the measurement of IFNAR2 expression in PBMCs may be useful for monitoring its expression in liver.

417–2α-Tocopherol and ascorbic acid attenuates the ribavirin-induced decrease of eicosapentaenoic acid in erythrocyte membrane in chronic hepatitis C patients

Background:  Oxidative damage of the erythrocyte membrane plays an important role in ribavirin‐induced anemia. The purpose of the present paper was to assess whether supplementation of α‐tocopherol and ascorbic acid (vitamins) causes changes in the erythrocyte membrane fatty acid composition during interferon and ribavirin combination therapy for chronic hepatitis C patients.

TYPE I INTERFERON RECEPTOR AND RESPONSE TO INTERFERON THERAPY IN CHRONIC HEPATITIS C PATIENTS: A PROSPECTIVE STUDY

Summary.  The type I interferon (IFN) receptor consists of at least two subunits, IFNAR1 and IFNAR2. We previously found a correlation between IFNAR1 and IFNAR2 expression in liver, and a correlation in IFNAR2 expression, but not in IFNAR1, between liver and peripheral blood mononuclear cells (PBMCs). The aim of this study was to prospectively assess whether IFNAR2 expression levels in PBMCs as well as in liver act as markers for predicting response to IFN therapy in chronic hepatitis C patients. Fifty‐two Japanese patients with chronic hepatitis C, were enrolled. IFNAR2 mRNA was quantified using competitive polymerase chain reaction, in liver and PBMC specimens, and of the 52 patients assigned to receive a 6‐month course of interferon‐α therapy, 36 patients who received more than 300 million units of interferon were analysed. IFNAR2 mRNA expression levels were significantly higher in liver than in PBMCs in all 36 patients (P = 0.016). Seventeen sustained virologic responders showed lower pretreatment hepatitis C virus (HCV)‐RNA levels (P = 0.017) in serum and higher pretreatment levels of IFNAR2 mRNA in liver (P = 0.007), but not in PBMCs, compared with nonsustained virologic responders. In multivariate analysis, these factors were independently associated with a sustained virologic response (i.e. HCV‐RNA level: odds ratio 0.23, 95% CI 0.038–0.864; and IFNAR2 in liver: odds ratio 1.116, 95% CI 1.015–1.227). Hence, IFNAR2 expression levels in liver, but not in PBMCs, is predictive of response to IFN treatment in chronic hepatitis C patients.

A possible role of hypervariable region 1 quasispecies in escape of hepatitis C virus particles from neutralization

We examined serial changes in the hypervariable region 1(HVR1) quasispecies both in immune and nonimmune complexed hepatitis C virus (HCV) particles from 12 patients with chronic hepatitis C to elucidate the mechanism by which genetic diversification of HCV during the course of infection allows escape of virus from the humoural immune response. Immune and nonimmune complexes were separated by differential flotation centrifugation and immunoprecipitation, and their HVR1 quasispecies were determined by subcloning and sequencing. The presence of a specific antibody against a specific viral clone in serum was examined in two patients by Western blotting of the corresponding recombinant HVR1 protein. The distribution of HVR1 quasispecies in both immune and nonimmune complexes conspicuously changed over time in most of the patients studied. In seven patients, various HCV clones serially shifted from nonimmune complexes to immune complexes. In four of them, a group of clones with similar HVR1 sequences to each other remained predominant in nonimmune complexes, whereas minor clones with sequences considerably divergent from the predominant clones shifted from nonimmune complexes to immune complexes. These results suggest a mechanism for persistent infection of HCV, in which major HCV clones escape from neutralization by anti‐HVR1 antibodies by generating considerably divergent minor ‘decoy’ clones which may be preferentially neutralized.

Constrained genomic and conformational variability of the hypervariable region 1 of hepatitis C virus in chronically infected patients.

We analysed the genomic and conformational variability of the hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) to evaluate the importance of its biological role. A total of 865 genotype 1b HVR1 subclones were collected from serially sampled sera in 11 patients with chronic hepatitis C, four of whom received interferon therapy. Consequently, 169 distinct sequences were examined for amino acid substitutions as well as hydrophilic or hydrophobic profile at each amino acid position within HVR1. Secondary structure of HVR1 was also predicted by the method of Robson in 90 distinct sequences from eight patients, including three interferon‐treated patients. Some positions within the HVR1 were invariable or nearly so as to amino acid substitution. Hydrophilic or hydrophobic residues exclusively predominated at several positions. These constrained amino acid replacement and hydrophilic or hydrophobic profiles were conserved irrespective of interferon therapy, though the frequency of amino acid replacement was greater at almost all amino acid positions within the HVR1 in interferon‐treated patients. The quasispecies of HCV showed various secondary structures of HVR1, but many sequences seemed to have common characteristics. β sheet conformations around both the N‐terminus and position 20 (numbered from the NH2 terminus of E2 envelope glycoprotein), and/or coil structures around the C‐terminus of HVR1 could be identified. These results suggest that HVR1 amino acid replacements are strongly constrained by a well‐ordered structure, in spite of being tolerant to amino acid substitutions, and imply an important biological role of the HVR1 protein in HCV replication.

Pilot study of combination therapy with transcatheter arterial infusion chemotherapy using iodized oil and percutaneous radiofrequency ablation during occlusion of hepatic blood flow for hepatocellular carcinoma.

Objective:We have reported that radiofrequency (RF) ablation with balloon occlusion of the hepatic artery (balloon-occluded RF ablation) increases the coagulation area compared with standard RF ablation. In this study, we evaluated the efficacy and safety of combination therapy with transcatheter arterial infusion chemotherapy (TAI) using iodized oil and balloon-occluded RF ablation in patients with hepatocellular carcinoma. Patients and Methods:We studied 12 patients with 12 HCC nodules (mean tumor diameter, 27.3 mm). All patients were classified as Child-Pugh Class A. Immediately after TAI using iodized oil, we performed balloon-occluded RF ablation. Results:One treatment session of the combination therapy was done for 10 of 12 nodules (83%). The greatest long-axis and short-axis dimensions of the area coagulated after the combination therapy were 48.8± 5.5 mm and 41.9 ± 4.1 mm, respectively. During follow-up (mean, 33.4 months), there was no local recurrence. The 1, 2, and 3-year survival rates were 100%, 92%, and 83%, respectively. No fatal complications were observed. Conclusions:The combination therapy is an effective and safe treatment under favorable liver reserve capacity. Using the combination therapy, it is possible to finish one treatment session for patients with HCC nodules measuring less than 3 cm in greatest dimension.

Hepatic expression of type I interferon receptor for predicting response to interferon therapy in chronic hepatitis C patients: a comparison of immunohistochemical method vs. competitive polymerase chain reaction assay

The focus of this study is to determine both mRNA and protein expression levels of the type I interferon (IFN) receptor subunits, IFNalpha receptor (IFNAR1) and IFNalpha/beta receptor (IFNAR2), in the liver, and to assess which quantification method is most useful in predicting response to IFN in chronic hepatitis C patients. Liver biopsy specimens from 41 chronic hepatitis C patients subsequently treated with IFN were immunostained with IFNAR1 and IFNAR2 antibodies, and also analyzed for both receptor subunit mRNA levels, using competitive polymerase chain reaction. Immunostaining of IFNAR1 and IFNAR2 was observed in the cell membrane and cytoplasm of hepatocytes in all liver specimens. Hepatic expression of IFNAR1 (r=0.45, P=0.012) or IFNAR2 mRNA (r=0.46, P=0.009) was weakly correlated with their protein expression in hepatocytes. The labeling indexes of IFNAR1 (136.7+/-94.1 vs. 77.4+/-76.8, P=0.032) and IFNAR2 (153.1+/-80.2 vs. 87.2+/-75.5, P=0.011) in liver specimens were significantly higher in sustained virologic responders (n=17) than in non-sustained virologic responders (n=24), while mRNA levels of either receptor subunit did not differ between response groups. These results suggest that protein expression of the type I IFN receptor in liver is a more useful measure than its mRNA expression in predicting response to IFN in chronic hepatitis C patients.

Fulminant hepatitis complicated by small intestine infection and massive hemorrhage

Abstract: A 34-year-old man diagnosed with fulminant hepatitis, caused by hepatitis B virus, and acute renal failure was referred to our hospital. After admission to the intensive care unit, the liver and renal failure were ameliorated. Melena requiring transfusion occurred during the course of his illness. Endoscopic examination demonstrated pseudomembranes, erosions, ulcers, and hemorrhage in the duodenum, the upper jejunum, and the terminal ileum, suggesting widespread lesions throughout the small intestine. Pseudomonas putida, Xanthomonas maltophilia, and Candida glabrata were cultured from ileal fluid. Candida glabrata was also detected in sputum, feces, and on an intravenous catheter tip. The patient was treated with amphotericin B and miconazole. The melena was ameliorated, but inflammation of the small intestine persisted. Although we had difficulty in treating the enteritis, the patient survived, and 1 year later colonoscopic examination demonstrated no abnormalities. The small intestine is a difficult site to examine, but endoscopic examination of this site is important when massive hemorrhage develops.