Isao Sakaida

Genome-wide association of IL28B with response to pegylated interferon-α and ribavirin therapy for chronic hepatitis C

The recommended treatment for patients with chronic hepatitis C, pegylated interferon-α (PEG-IFN-α) plus ribavirin (RBV), does not provide sustained virologic response (SVR) in all patients. We report a genome-wide association study (GWAS) to null virological response (NVR) in the treatment of patients with hepatitis C virus (HCV) genotype 1 within a Japanese population. We found two SNPs near the gene IL28B on chromosome 19 to be strongly associated with NVR (rs12980275, P = 1.93 × 10−13, and rs8099917, 3.11 × 10−15). We replicated these associations in an independent cohort (combined P values, 2.84 × 10−27 (OR = 17.7; 95% CI = 10.0–31.3) and 2.68 × 10−32 (OR = 27.1; 95% CI = 14.6–50.3), respectively). Compared to NVR, these SNPs were also associated with SVR (rs12980275, P = 3.99 × 10−24, and rs8099917, P = 1.11 × 10−27). In further fine mapping of the region, seven SNPs (rs8105790, rs11881222, rs8103142, rs28416813, rs4803219, rs8099917 and rs7248668) located in the IL28B region showed the most significant associations (P = 5.52 × 10−28–2.68 × 10−32; OR = 22.3–27.1). Real-time quantitative PCR assays in peripheral blood mononuclear cells showed lower IL28B expression levels in individuals carrying the minor alleles (P = 0.015).

Transplantation of bone marrow cells reduces CCl4‐induced liver fibrosis in mice

We investigated the effect of bone marrow cell (BMC) transplantation on established liver fibrosis. BMCs of green fluorescent protein (GFP) mice were transplanted into 4‐week carbon tetrachloride (CCl4)–treated C57BL6 mice through the tail vein, and the mice were treated for 4 more weeks with CCl4 (total, 8 weeks). Sirius red and GFP staining clearly indicated migrated BMCs existing along with fibers, with strong expression of matrix metalloproteinase (MMP)‐9 shown by anti–MMP‐9 antibodies and in situ hybridization. Double fluorescent immunohistochemistry showed the expression of MMP‐9 on the GFP‐positive cell surface. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area coinciding with the location of MMP‐9–positive BMCs. Four weeks after BMC transplantation, mice had significantly reduced liver fibrosis, as assessed by hydroxyproline content of the livers, compared to that of mice treated with CCl4 alone. Subpopulation of Liv8‐negative BMCs was responsible for this fibrolytic effect. In conclusion, mice with BMC transplants with continuous CCl4 injection had reduced liver fibrosis and a significantly improved survival rate after BMC transplantation compared with mice treated with CCl4 alone. This finding introduces a new concept for the therapy of liver fibrosis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:1304–1311.)

Improved Liver Function in Patients with Liver Cirrhosis After Autologous Bone Marrow Cell Infusion Therapy

We here report nine liver cirrhosis (LC) patients that underwent autologous bone marrow cell infusion (ABMI) from the peripheral vein. Subjects were patients with LC with total bilirubin of less than 3.0 mg/dl, platelet count of more than 5 (1010/l), and no viable hepatocellular carcinoma on diagnostic imaging. Autologous bone marrow (BM; 400 ml) was isolated from the ilium under general anesthesia. Mononuclear cells (MNCs) were separated by cell washing and were infused via the peripheral vein. MNC characteristics were confirmed by fluorescence‐activated cell sorting analysis (CD34, CD45, and c‐kit). After ABMI therapy, liver function was monitored by blood examination for 24 weeks. From 400 ml of BM, we obtained 7.81 ± 0.98 × 109 MNCs. After washing, 5.20 ± 0.63 × 109 MNCs were infused into patients with LC. Significant improvements in serum albumin levels and total protein were observed at 24 weeks after ABMI therapy (p < .05). Significantly improved Child‐Pugh scores were seen at 4 and 24 weeks (p < .05). α‐Fetoprotein and proliferating cell nuclear antigen (PCNA) expression in liver biopsy tissue was significantly elevated after ABMI therapy (p < .05). No major adverse effects were noted. In conclusion, ABMI therapy should be considered as a novel treatment for patients with decompensated LC.

Hepatitis C Virus–Induced Reactive Oxygen Species Raise Hepatic Iron Level in Mice by Reducing Hepcidin Transcription

BACKGROUND & AIMS Despite abundant clinical evidence, the mechanisms by which hepatic iron overload develops in patients with hepatitis C virus (HCV)-associated chronic liver disease remain unknown. The aim of this study was to investigate how hepatic iron overload develops in the presence of HCV proteins. METHODS Male transgenic mice expressing the HCV polyprotein and nontransgenic control mice (C57BL/6) were assessed for iron concentrations in the liver, spleen, and serum and iron regulatory molecules in vivo and ex vivo. RESULTS Transgenic mice had increased hepatic and serum iron concentrations, decreased splenic iron concentration, and lower hepcidin expression in the liver accompanied by higher expression of ferroportin in the duodenum, spleen, and liver. In response to hepatocellular iron excess, transferrin receptor 1 expression decreased and ferritin expression increased in the transgenic liver. Transgenic mice showed no inflammation in the liver but preserved the ability to induce hepcidin in response to proinflammatory cytokines induced by lipopolysaccharide. Hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein alpha (C/EBP) were down-regulated concomitant with increased expression of C/EBP homology protein, an inhibitor of C/EBP DNA binding activity, and with increased levels of reactive oxygen species in transgenic mice at the ages of 8 and 14 months. CONCLUSIONS HCV-induced reactive oxygen species may down-regulate hepcidin transcription through inhibition of C/EBPalpha DNA binding activity by C/EBP homology protein, which in turn leads to increased duodenal iron transport and macrophage iron release, causing hepatic iron accumulation.

Herbal medicine Inchin-ko-to (TJ-135) prevents liver fibrosis and enzyme-altered lesions in rat liver cirrhosis induced by a choline-deficient l-amino acid-defined diet

BACKGROUND/AIMS The herbal medicine Inchin-ko-to (TJ-135), extract power from three herbs, has recently been reported possessing anti-apoptotic activity. The aim of this study was to investigate whether TJ-135 has any influence on the development of preneoplastic lesions as well as liver fibrosis. METHODS The effects of the TJ-135 were examined using the choline-deficient L-amino acid-defined diet-induced liver fibrosis model. In addition, the effect of TJ-135 on mitogen-activated protein (MAP) kinase, type III procollagen mRNA expression and the medium N-terminal procollagen III propeptide (PIIINP) concentration in a hepatic stellate cell line (LI90) were examined. RESULTS TJ-135 prevented fibrosis in a dose-dependent manner up to 1.5% (w/w). TJ-135 also reduced the expression of type III procollagen mRNA in the liver, as well as the number of activated stellate cells. Furthermore, TJ-135 reduced the area of preneoplastic lesions in the liver. With LI90 cells, TJ-135 reduced MAP kinase (ERK and JNK but not P38) activities resulting in reduced type III procollagen mRNA and PIIINP concentrations in the medium in a dose-dependent manner. CONCLUSIONS These results indicate that although TJ-135 has anti-apoptotic activity, TJ-135 does not increase preneoplastic lesions but significantly reduces liver fibrosis through the inhibition of stellate cell activation without a reduction of hepatocyte cell death.

General rules for recording endoscopic findings of esophagogastric varices (2ND EDITION)

General rules for recording endoscopic findings of esophageal varices were initially proposed in 1980 and revised in 1991. These rules have widely been used in Japan and other countries. Recently, portal hypertensive gastropathy has been recognized as a distinct histological and functional entity. Endoscopic ultrasonography can clearly depict vascular structures around the esophageal wall in patients with portal hypertension. Owing to progress in medicine, we have updated and slightly modified the former rules. The revised rules are simpler and more straightforward than the former rules and include newly recognized findings of portal hypertensive gastropathy and a new classification for endoscopic ultrasonographic findings.

Genome-Wide Association Study Confirming Association of HLA-DP with Protection against Chronic Hepatitis B and Viral Clearance in Japanese and Korean

Hepatitis B virus (HBV) infection can lead to serious liver diseases, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC); however, about 85–90% of infected individuals become inactive carriers with sustained biochemical remission and very low risk of LC or HCC. To identify host genetic factors contributing to HBV clearance, we conducted genome-wide association studies (GWAS) and replication analysis using samples from HBV carriers and spontaneously HBV-resolved Japanese and Korean individuals. Association analysis in the Japanese and Korean data identified the HLA-DPA1 and HLA-DPB1 genes with Pmeta = 1.89×10−12 for rs3077 and Pmeta = 9.69×10−10 for rs9277542. We also found that the HLA-DPA1 and HLA-DPB1 genes were significantly associated with protective effects against chronic hepatitis B (CHB) in Japanese, Korean and other Asian populations, including Chinese and Thai individuals (Pmeta = 4.40×10−19 for rs3077 and Pmeta = 1.28×10−15 for rs9277542). These results suggest that the associations between the HLA-DP locus and the protective effects against persistent HBV infection and with clearance of HBV were replicated widely in East Asian populations; however, there are no reports of GWAS in Caucasian or African populations. Based on the GWAS in this study, there were no significant SNPs associated with HCC development. To clarify the pathogenesis of CHB and the mechanisms of HBV clearance, further studies are necessary, including functional analyses of the HLA-DP molecule.

The efficacy of ursodeoxycholic acid and bezafibrate combination therapy for primary biliary cirrhosis: A prospective, multicenter study

Aim:  Treatment with ursodeoxycholic acid (UDCA) improves the survival of stage I and II primary biliary cirrhosis (PBC) patients. However, new therapeutic options are needed for patients who are refractory to UDCA and for those whose disease is at an advanced stage. Bezafibrate could be useful in PBC treatment, since it increases phospholipid output into the bile and reduces the cytotoxicity of hydrophobic bile acids, which are increased with cholestasis.

Inhibitory effects of the herbal medicine Sho-saiko-to (TJ-9) on cell proliferation and procollagen gene expressions in cultured rat hepatic stellate cells

BACKGROUND/AIMS It is of extreme importance to prevent liver fibrosis and subsequent progression to liver cirrhosis. The aim of our study was to elucidate in vitro whether Sho-saiko-to exerted inhibitory effects on hepatic stellate cells. METHODS Hepatic stellate cells were isolated from male Wistar rats. Water-soluble ingredients of Sho-saiko-to were obtained at concentrations of 10, 100, 250, 500 and 1000 microg/ml. Morphological transformation was observed under a phase-contrast microscope. Flow cytometric analysis was performed on day 4 after culture to evaluate the potential to proliferate of the stellate cells by analyzing cell cycles. Northern blot analysis was carried out on day 3 after culture to determine the expressions of type I and type III procollagen mRNAs. RESULTS (i) Sho-saiko-to 500 and 1000 microg/ml inhibited morphological transformation of the stellate cells to myofibroblast-like cells. (ii) Sho-saiko-to 500 and 1000 microg/ml significantly (p<0.0001) accumulated the cells in the G0/G1 phase (118.8+/-0.7%, 119.2+/-0.5%, respectively as compared with control) and significantly (p<0.0001) decreased cell numbers subsequently in G2/M phase (47.5+/-8.1%, 48.9+/-2.0%, respectively). (iii) Sho-saiko-to 500 and 1000 microg/ml also significantly (p<0.05 or p<0.0001) suppressed procollagen mRNA expression of type I to 51.5+/-6.4%, 34.9+/-3.7%, respectively, and type III to 51.3+/-12.3%, 46.7+/-11.4%, respectively. CONCLUSIONS We have clarified the inhibitory effects of Sho-saiko-to on hepatic stellate cells in vitro. Sho-saiko-to could be a potent inhibitor in the pathogenesis of liver fibrosis.

Tolvaptan for improvement of hepatic edema: A phase 3, multicenter, randomized, double‐blind, placebo‐controlled trial

Hepatic edema is manifested by ascites, lower limb edema and intolerable symptoms. Some patients insufficiently respond to the conventional diuretic therapy. Therefore, a novel therapeutic option is required. We conducted a phase 3 study to confirm therapeutic effect of tolvaptan on hepatic edema associated with liver cirrhosis.