Atsushi Hokari

Inducible Nitric Oxide Synthase in a Human Glioblastoma Cell Line

Abstract: Nitric oxide synthase (NOS) activity was induced in the cytosol of A‐172 cells by treatment with lipopolysaccharide, tumor necrosis factor‐α, and interferon‐γ. A 130‐kDa human inducible NOS (iNOS) protein was detected with anti‐rat iNOS antibody by western blot analysis. Northern blot analysis showed that the iNOS mRNA was ∼4.5 kb, using a cDNA fragment for human iNOS, isolated from stimulated A‐172 cells by reverse transcriptase‐PCR, as a probe. The mRNA was induced by interferon‐γ at a trace level, and its expression was synergistically enhanced by simultaneous addition of lipopolysaccharide, tumor necrosis factor‐α, and, to a larger extent, interleukin‐1β. The mRNA expression was blocked by coincubation with actinomycin D or cycloheximide. Furthermore, by transfecting the mouse iNOS gene promoter into A‐172 cells, transcriptional activation of the iNOS gene was detected in these cells upon stimulation with lipopolysaccharide and cytokines. The pattern of promoter activation correlated well with that of iNOS mRNA expression upon stimulation. These data indicate that expression of iNOS is transcriptionally regulated in A‐172 cells. This process requires de novo protein synthesis with a mechanism similar to that in place in mouse macrophages.

Intrahepatic expression of the co-stimulatory molecules programmed death-1, and its ligands in autoimmune liver disease.

Liver‐infiltrating T cells play an essential role in the immunopathogenesis of autoimmune liver disease. Programmed death‐1 (PD‐1) and its ligands, B7‐H1/PD‐L1 and B7‐DC/PD‐L2, are new CD28‐B7 family members that are involved in the regulation of immune responses. The ligation of PD‐1 inhibits T‐cell receptor‐mediated T cell proliferation and cytokine production, and PD‐1‐deficient mice develop various organ‐specific autoimmune diseases. To investigate the expressions of PD‐1 and its ligands in autoimmune liver disease, in particular autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC), immunohistochemical analysis was performed. Liver biopsy specimens obtained from 17 patients with AIH and PBC were studied. PD‐1 was expressed on more than half of the liver‐infiltrating T cells within the portal tract. Some of the intrahepatic T cells expressed B7‐H1 in patients with AIH and PBC. B7‐H1 and B7‐DC were mainly expressed on some Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC) within the sinusoids and their expression was upregulated in autoimmune liver disease. These results suggest that the interaction of PD‐1 on T cells with increased expression of B7‐H1 and B7‐DC on KC and LSEC might be involved in the downregulation of autoreactive lymphocytes and result in the regulation of pathogenesis in autoimmune liver disease.

Detection of serum nitrite and nitrate in primary biliary cirrhosis: Possible role of nitric oxide in bile duct injury

Background: The role of nitric oxide synthase (NOS) in autoimmune disease is gaining increased attention because of the relationships between NOS activity and T‐lymphocyte subpopulations and, in particular, the influence of NO on cytokine production by Th1 versus Th2 cells. In addition, there is evidence that both the liver and infiltrating hepatic T cells have inducible NOS‐2 activity.

Autoimmune hepatitis: current challenges and future prospects

Autoimmune hepatitis (AIH) is a chronic progressive liver disease characterized by high levels of aminotransferases and autoantibodies, hypergammaglobulinemia, and interface hepatitis. AIH affects all races and all ages worldwide, regardless of sex, although a preponderance of females is a constant finding. The etiology of AIH has not been completely elucidated, but immunogenetic background and environmental parameters may contribute to its development. The most important genetic factor is human leukocyte antigens (HLAs), especially HLA-DR, whereas the role of environmental factors is not completely understood. Immunologically, disruption of the immune tolerance to autologous liver antigens may be a trigger of AIH. The diagnosis of classical AIH is fairly easy, though not without pitfalls. In contrast, the diagnosis of atypical AIH poses great challenges. There is confusion as to the definition of the disease entity and its boundaries in the diagnosis of overlap syndrome, drug-induced autoimmune hepatitis, and AIH with concomitant nonalcoholic fatty liver disease (NAFLD) or chronic hepatitis C. Centrilobular zonal necrosis is now included in the histological spectrum of AIH. However, the definition and the significance of AIH presenting with centrilobular zonal necrosis have not been examined extensively. In ~20% of AIH patients who are treated for the first time with standard therapy, remission is not achieved. The development of more effective and better tolerated novel therapies is an urgent need. In this review, we discuss the current challenges and the future prospects in relation to the diagnosis and treatment of AIH, which have been attracting considerable recent attention.

Implication of nitric oxide synthase in carcinogenesis: analysis of the human inducible nitric oxide synthase gene

Nitric oxide (NO) is a newly identified, multifunctional biological mediator. However, it also has deleterious effects on biological materials. For instance, nucleic acids, proteins, and some prosthetic groups of enzymes can be modified by NO or its reaction products with other reactive oxygen species. Endogenous nitrosamine formation through the reaction of NO or its oxidized products with amines might be involved in carcinogenesis. These deleterious effects of NO are often associated with inflammatory processes both in experimental animals and human. We analyzed the molecular mechanism of control of expression of the inducible nitric oxide synthase (NOS) gene in mouse cells by cloning its putative promoter region. This promoter responded to various cytokines and endotoxin similarly to the endogenous NOS gene in mouse cells. No appreciable induction of NOS was observed in human peripheral blood cells, but induction was detected in a human glioblastoma cell line A-172. Therefore, the human inducible NOS cDNA was cloned from A-172 cells and its cDNA-deduced amino acid sequence found to have about 80% similarity to those of both mouse and rat inducible NOSs. The effects of various cytokines on the induction of the gene were somewhat different from those observed in mouse cells, but the mouse promoter responded to these cytokines similarly to the endogenous NOS gene in human cells, indicating functional similarity of cis-elements of the genes encoding both human and mouse inducible NOS. Structural analysis of the human inducible NOS gene by Southern blot analysis revealed putative genetic restriction fragment length polymorphism in intron 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Impact of interferon-free antivirus therapy on lipid profiles in patients with chronic hepatitis C genotype 1b

AIM To investigate the influence of interferon-free antivirus therapy on lipid profiles in chronic hepatitis C virus genotype 1b (HCV1b) infection. METHODS Interferon-free antiviral agents were used to treat 276 patients with chronic HCV1b infection, and changes in serum lipids of those who achieved sustained virologic response (SVR) were examined. The treatment regimen included 24 wk of daclatasvir plus asunaprevir (DCV + ASV) or 12 wk of sofosbuvir plus ledipasvir (SOF + LDV). SVR was achieved in 121 (85.8%) of 141 patients treated with DCV + ASV and 132 (97.8%) of 135 patients treated with SOF + LDV. In the two patient groups (DCV + ASV-SVR and SOF + LDV-SVR), serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides were measured at baseline during treatment and at 4 and 12 wk after treatment. Then, longitudinal changes in lipid profiles were analyzed. RESULTS Serum levels of TC, LDL-C, and HDL-C were significantly increased throughout the observation period in both the DCV + ASV-SVR and SOF + LDV-SVR groups. During antivirus treatment, the increases in TC and LDL-C were significantly greater in the SOF + LDV-SVR group than in the DCV + ASV-SVR group (P < 0.001). At 4 and 12 wk after the therapy, serum levels of TC and LDL-C were similar between the two groups and were significantly greater than those at baseline. Approximately 75%-80% of the increase in TC was derived from an increased LDL-C. In multiple regression analysis, the difference in therapy protocol (DCA + ASV or SOF + LDV) was an independent predictor that was significantly associated with the increase in TC and LDL-C at 4 wk of therapy. CONCLUSION Serum cholesterol significantly increased during SOF + LDV treatment. After treatment, HCV elimination was associated with a similar increase in cholesterol regardless of the therapy protocol.

Efficacy of MK615 for the treatment of patients with liver disorders

AIM To investigate the hepatoprotective effect of MK615, a Japanese apricot extract, in an animal model, and its clinical therapeutic effect. METHODS Wistar rats were administered physiological saline (4 mL/kg) or MK615 solution (4 mL/kg) for 7 d. On the sixth d, acute hepatic injury was induced by administering a single intraperitoneal injection (ip) of D-galactosamine hydrochloride (D-GalN) (600 mg/kg). Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined, and liver tissues were used for histopathological analysis. Fifty-eight patients with liver disorders [hepatitis C (n = 40), non-alcoholic fatty liver disease (n = 15), and autoimmune liver disease (n = 3)] were orally administered commercially available Misatol ME-containing MK615 (13 g/d) daily for 12 wk. Blood and urine were sampled immediately before and 6 wk, 12 wk, and 16 wk after the start of intake to measure various biochemical parameters. The percentage change in ALT and AST levels after 12 wk from the pre-intake baseline served as a primary endpoint. RESULTS D-GalN effectively induced acute hepatic injury in the rats. At 48 h after the ip injection of D-GalN, the plasma levels of ALT (475.6 ± 191.5 IU/L vs 225.3 ± 194.2 IU/L, P < 0.05) and AST (1253.9 ± 223.4 IU/L vs 621.9 ± 478.2 IU/L, P < 0.05) in the MK615 group were significantly lower than the control group. Scattered single cell necrosis, loss of hepatocytes, and extensive inflammatory cell infiltration were observed in hepatic tissue samples collected from the control group. However, these findings were less pronounced in the group receiving MK615. At the end of the clinical study, serum ALT and AST levels were significantly decreased compared with pre-intake baseline levels from 103.5 ± 58.8 IU/L to 71.8 ± 39.3 IU/L (P < 0.05) and from 93.5 ± 55.6 IU/L to 65.5 ± 34.8 IU/L (P < 0.05), respectively. A reduction of ≥ 30% from the pre-study baseline ALT level was observed in 26 (45%) of the 58 patients, while 25 (43%) patients exhibited similar AST level reductions. The chronic hepatitis C group exhibited significant ALT and AST level reductions from 93.4 ± 51.1 IU/L to 64.6 ± 35.1 IU/L (P < 0.05) and from 94.2 ± 55.5 IU/L to 67.2 ± 35.6 IU/L (P < 0.05), respectively. A reduction of ≥ 30% from the pre-study baseline ALT level was observed in 20 (50%) of the 40 patients. ALT levels in both the combined ursodeoxycholic acid (UDCA) treatment and the UDCA uncombined groups were significantly lower after Misatol ME administration. MK615 protected hepatocytes from D-GalN-induced cytotoxicity in rats. Misatol ME decreased elevated ALT and AST levels in patients with liver disorders. CONCLUSION These results suggest that MK615 and Misatol ME are promising hepatoprotective agents for patients with liver disorders.

Significance of interferon-β for the treatment of hepatitis C virus infection in hemodialyzed patients.

Aim:  We evaluated the adverse effects and efficacy associated with interferon‐β (IFN‐β) for the treatment of hepatitis C virus (HCV) infection in 20 hemodialyzed (HD) patients.

Clinical usefulness of ursodeoxycholic acid for Japanese patients with autoimmune hepatitis

AIM To evaluate the therapeutic effects of ursodeoxycholic acid (UDCA) on autoimmune hepatitis (AIH). METHODS A total 136 patients who were diagnosed with AIH were included in our study. All of the patients underwent a liver biopsy, and had at least a probable diagnosis on the basis of either the revised scoring system or the simplified scores. Initial treatment included UDCA monotherapy (Group U, n = 48) and prednisolone (PSL) monotherapy (Group P, n = 88). Group U was further classified into two subgroups according to the effect of UDCA: Patients who had achieved remission induction with UDCA monotherapy and showed no sign of relapse (Subgroup U1, n = 34) and patients who additionally received PSL during follow-up (Subgroup U2, n = 14). We compared the clinical and histological findings between each groups, and investigated factors contributing to the response to UDCA monotherapy. RESULTS In Group U, 34 patients (71%) achieved and maintained remission over 49 (range: 8-90) mo (Subgroup U1) and 14 patients (29%) additionally received PSL (Subgroup U2) during follow-up. Two patients in Subgroup U2 achieved remission induction once but additionally required PSL administration because of relapse (15 and 35 mo after the start of treatment). The remaining 12 patients in Subgroup U2 failed to achieve remission induction during follow-up, and PSL was added during 7 (range: 2-18) mo. Compared with Subgroup U2, Subgroup U1 had significantly lower alanine aminotransferase (ALT) levels at onset (124 IU/L vs 262 IU/L, P = 0.023) and a significantly higher proportion of patients with mild inflammation (A1) on histological examination (70.6% vs 35.7%, P = 0.025). When multivariate analysis was performed to identify factors contributing to the response to UDCA monotherapy, only a serum ALT level of 200 IU/L or lower was found to be associated with a significant difference (P = 0.013). CONCLUSION To prevent adverse events related to corticosteroids, UDCA monotherapy for AIH needs to be considered in patients with a serum ALT level of 200 IU/L or lower.

A case of autoimmune hepatitis diagnosed as liver cirrhosis after 13 yeares following uup without corticosteroid therapy

症例は66歳女性. 1984年に全身倦怠感を主訴に入院. 血液検査および肝生検により自己免疫性肝炎 (Autoimmune hepatitis: AIH) が疑われた. ALT上昇は軽度で安静により自覚症状が改善したため, 患者は副腎皮質ステロイド治療を希望せず, その後医療機関を受診しなかった. 1994年に再受診した際にALTの上昇を認め, ウルソデオキシコール酸 (UDCA) 投与によりALTは持続的に改善したが, 1997年に施行した肝生検で高度な活動性を伴う肝硬変への進展が確認された. 無治療のAIH症例の長期予後を検討し得た報告は極めて稀であり, ALT上昇が軽度で無症状のAIH症例の治療法を考慮するうえで示唆に富む症例と考えられる.