Makoto Yoshiba

Fulminant hepatitis B: Induction by hepatitis B virus mutants defective in the precore region and incapable of encoding E antigen

Clones of hepatitis B virus were propagated from 10 cases of fulminant hepatitis B after amplification by polymerase chain reaction and their nucleotide sequences of the precore region were determined. All 113 clones from 9 cases had a point mutation from guanine to adenine at nucleotide 83 in the precore region, which converted codon 28 for tryptophan (TGG) to a stop codon (TAG) and prohibited the synthesis and secretion of hepatitis B e antigen. Precore-region defects were not detected in any of 23 clones from the remaining 1 case. By contrast, precore-region defects were not found in any of 180 clones from 8 cases of acute hepatitis B without hepatic failure serving as controls. The source of infection was traceable in 3 cases. The same precore-region defect, along with the sequence identity of 435 nucleotides, was observed in clones from the case of a baby and his grandmother, who carried the virus and was implicated in the transmission, and also in clones from two pediatricians and the carrier patients they attended. These findings support the hypothesis that precore-defective mutants have stronger activity to induce fulminant hepatitis than nondefective viruses.

Combined interferon α2b and cyclosporin A in the treatment of chronic hepatitis C: controlled trial

Background. Only 15% to 20% of patients with chronic hepatitis C have a sustained virological response to interferon monotherapy. The aim of the present study was to compare the efficacy and safety of interferon, in combination with oral cyclosporin A, with interferon monotherapy in the treatment of chronic hepatitis C. Methods. We assigned 120 patients with chronic hepatitis C to receive the standard Japanese dose of interferon α2b alone for 24 weeks or that dose of interferon α2b in combination with cyclosporin A, at doses of 200 mg daily for the first 4 weeks and 100 mg daily for the following 20 weeks. All patients were assessed for drug safety, tolerance, and efficacy at the end of weeks 4, 12, 24, and 48. Efficacy was assessed by the disappearance of serum hepatitis C virus (HCV)-RNA by polymerase chain reaction and normalization of serum aminotransferase. The primary endpoint was a sustained virological response; i.e., sustained undetectable serum HCV RNA at 48 weeks. Results. The sustained virological response rate was significantly higher in the combination therapy group (42/76) than in the monotherapy group (14/44; P = 0.01). The sustained biochemical response rate was also higher in the combination therapy group (46/76) than in the monotherapy group (17/44; P = 0.017). In patients with genotype 1 and high viral loads, the sustained virological response rate was markedly higher in the combination therapy group (16/38) than in the monotherapy group (1/21; P = 0.006). Side-effect profiles were similar in the two groups. Conclusions. In patients with chronic hepatitis C; combined interferon and cyclosporin A treatment was more effective than interferon monotherapy. The benefit was mostly achieved in patients with a high viral load and HCV genotype 1.

Evaluation of a cyclophilin inhibitor in hepatitis C virus-infected chimeric mice in vivo.

Cyclosporin A (CsA) inhibits replication of the HCV subgenomic replicon, and this effect is believed to not be mediated by its immunosuppressive action. We found that DEBIO‐025, a novel non‐immunosuppressive cyclophilin inhibitor derived from CsA, inhibited HCV replication in vitro more potently than CsA. We also examined the inhibitory effect of DEBIO‐025 on naive HCV genotypes 1a or 1b in vivo using chimeric mice with human hepatocytes. These mice were treated for 14 days with DEBIO‐025, pegylated‐interferon α−2a (Peg‐IFN), a combination of either drugs, or CsA in combination with Peg‐IFN. In mice treated with Peg‐IFN, serum HCV RNA levels decreased approximately 10‐fold whereas DEBIO‐025 treatment alone did not induce any significant change. In mice treated with both DEBIO‐025 and Peg‐IFN, HCV RNA levels decreased more than 100‐fold. All mice treated with Peg‐IFN combined with CsA died within 4 days. The combination treatment of DEBIO‐025 and Peg‐IFN reduced HCV RNA levels and core protein expression in liver, indicating that the HCV RNA levels reduction in serum was attributable to intrahepatic inhibition of HCV replication. Conclusion: We demonstrated that DEBIO‐025 was better tolerated than CsA, and that its anti‐HCV effect appeared to be synergistic in combination with Peg‐IFN in vivo. (HEPATOLOGY 2007;45:921–928.)

Development of reliable artificial liver support (ALS)--plasma exchange in combination with hemodiafiltration using high-performance membranes.

A new artificial liver support system (ALSS) consisting of plasma exchange (PE) in combination with hemodiafiltration (HDF) using high-performance membranes of polymethyl metacrylate (PMMA) and cellulose triacetate (CTA) was developed to efficiently remove middle molecules from plasma and treat fulminant hepatic failure (FHF) complicated, by the onset of hepatic coma. Twenty-seven patients with FHF due to viral hepatitis, two with type A (HA), nine with type B (HB), and 16 with type non-A, non-B (NANB) underwent therapy with this new ALSS over the last five years. Three patients, with an exacerbation of chronic HB and 15/16 with type NANB hepatitis were treated with interferon (IFN) also. Of these, 25 patients (92.6%), regained consciousness and 15 (55.6%) [1/2 (50%) with type A, 6/9 (66.7%) with type B and 8/16 (50%) with type NANB hepatitis] survived Including four patients who survived with intensive, care and plasma exchange alone, 19/31 (61.3%) patients survived. Because of its biocompatibility, both survivors and nonsurvivors could be sustained with the ALSS without complications for long periods (19.3 days for the survivors and 32.4 days for nonsurvivors). With this ALSS the ability to sustain life for such prolonged periods allows hepatic regeneration to occur and result in patient survival. It is anticipated that this new ALSS will not only be of value in cases of fulminant hepatic failure but that it may also play a role in sustaining life for those, awaiting liver transplantation.

Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype

RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.

Intraductal Tubular Neoplasms of the Pancreas : Histogenesis and Differentiation

Objectives: Intraductal neoplasms of the pancreas are generally referred to as intraductal papillary mucin-producing neoplasms (IPMNs), according to the WHO classification system. Herein, we report that morphologic and immunohistochemical features of intraductal tubular carcinoma (ITC) are quite different from those of intraductal papillary mucinous carcinoma (IPMC). Methods: We analyzed histogenesis and differentiation of ITC by light microscopy and immunohistochemistry. Results: Histologically, ITC was characterized as an intraductal nodular appearances with a monotonous tubular growth pattern without papillary projection. ITC showed de novo-like appearance without sequential progression usually observed in IPMC, suggesting that ITC is a homogeneous neoplasm. Cuboidal tumor cells in ITC resembled normal pancreatic duct epithelia, and the characteristic growth pattern of ITC replaced that of normal pancreatic duct epithelium. Immunohistochemically, ITC cells were positive for MUC-1 on the apical side of the cell membrane. In contrast to ITC cells, IPMC cells were negative for MUC-1, and ductal adenocarcinoma cells were strongly positive for MUC-1, as was the stroma around the cancer. The immunohistochemical staining pattern of DUPAN-2 resembled that of MUC-1. Interestingly, localization of MUC-1 and DUPAN-2 staining in ITC cells was similar to that in normal pancreatic ductules. ITC cells were negative for MUC-2 and MUC-5AC. In contrast, most IPMC cells were positive for MUC-2 and MUC-5AC. Conclusion: Based on our histologic and immunohistochemical findings, the intraductal pancreatic neoplasm (IPN) can be classified into 2 groups: IPN with gastrointestinal differentiation and IPN with pancreatic duct differentiation. Our present data indicated that ITC cells may arise directly from duct epithelia without progression and possessed pancreatic duct differentiation. On the basis of our data, we suggest that classification of pancreatic neoplasms in the WHO and The Armed Forces Institute of Pathology (AFIP) systems should be reconsidered.

The Presence of a Newly Identified Infectious Agent (SEN Virus) in Patients with Liver Diseases and in Blood Donors in Japan

The existence of the newly discovered SEN virus (SENV) was investigated in 379 Japanese patients with liver diseases and in 277 blood donors, to determine whether SENV is associated with non-A-E hepatitis. SENV DNA was detected by seminested polymerase chain reaction, with primers directed to 2 SENV strains: SENV-H and SENV-D. SENV was detected in 7 (32%) of 22 patients with fulminant hepatitis, in 15 (17%) of 86 patients with acute hepatitis, in 38 (27%) of 139 patients with chronic hepatitis, in 29 (31%) of 93 patients with liver cirrhosis, in 5 (33%) of 15 patients with autoimmune hepatitis, in 11 (46%) of 24 patients with primary biliary cirrhosis, and in 27 blood donors (10%). Infection occurred more frequently in patients with liver diseases than in blood donors; however, there were no significant differences in SENV-positive rates between patients with non-A-C hepatitis and those with acute or chronic hepatitis due to known hepatitis virus or nonviral liver disease. This study did not suggest SENV as a possible causative agent of non-A-C hepatitis.

Reactivation of precore mutant hepatitis B virus leading to fulminant hepatic failure following cytotoxic treatment

Three hepatitis B virus carriers who were HBeAg negative and having normal liver function developed fulminant hepatitis with evidence of HBV replication following intensive chemotherapy for non-Hodgkins lymphoma. Each was continuously negative for HBeAg. Analysis of the precore region of HBV isolated from each demonstrated that the HBV of each had a point mutation in the precore region that inhibited the synthesis and the release of hepatitis Be antigen. This observation suggests that all HB carriers receiving either immunosuppressive or cytotoxic therapy should be monitored closely even if standard assays suggest that viral replication is not present. Sudden enhanced replication of a HBV mutant as a result of such therapy can be a cause of either very severe hepatitis or occasionally fulminant hepatitis.

Histologic and Immunohistochemical Comparison of Intraductal Tubular Carcinoma, Intraductal Papillary-Mucinous Carcinoma, and Ductal Adenocarcinoma of the Pancreas

Objective: To analyze the differences between intraductal tubular carcinoma (ITC) and intraductal papillary-mucinous carcinoma (IPMC), we performed light microscopic and immunohistochemical analysis of 4 cases of ITC, 6 cases of IPMC, and 9 cases of ductal adenocarcinoma of the pancreas. Methods and Results: Light microscopic examination showed no hyperplasia or adenoma around the carcinoma in ITC, and immunohistochemical analysis showed that the apical side of the cell membrane was positive for MUC-1 in almost all ITC cells. In contrast to ITC cells, all IPMC cells were negative for MUC-1 and ductal adenocarcinoma cells were strongly positive for MUC-1 in the cytoplasm and cell membrane. Immunohistochemical staining patterns of DUPAN-2 in ITC resembled those of MUC-1 in these cancers. ITC and IPMC cells were negative for carcinoembryonic antigen, but ductal adenocarcinoma cells were positive. There were no apparent differences in proliferative activity between ITC and IPMC, but ductal adenocarcinoma showed significantly greater activity than either ITC or IPMC. Conclusion: The PCNA-L.I of IPMC and ITC was lower and the cell atypia of them was more mild compared with those of ductal carcinoma, indicating that IPMC possess low-grade malignant potentials. However, we observed differences of growth patterns and mucous secretion between ITC and IPMC of the pancreas.

Case Report: Fulminant Hepatitis C Viral Infection After Allogeneic Bone Marrow Transplantation

The authors describe two patients with acute leukemia who died of fulminant hepatitis caused by hepatitis C virus (HCV) after an allogeneic bone marrow transplant, the first such cases reported in Japan. Both had developed posttransfusion hepatitis during chemotherapy to induce remission and for consolidation. Six months after blood transfusion, the blood serum of each patient was positive for HCV antibody and HCV RNA. In each case, there was a transient improvement in liver function after the transplant. However, within 5 months of receiving the transplant and coincident with the withdrawal of cyclosporin A, each patient developed an acute exacerbation of hepatitis. The fulminant hepatitis in our patients may, therefore, have been caused by the reactivation of HCV induced by the immunosuppressive therapy followed by a reconstitution of the immune system.