Atsuhiro Morita

Increase of chemokine interferon‐inducible protein‐10 (IP‐10) in the serum of patients with autoimmune liver diseases and increase of its mRNA expression in hepatocytes

To clarify the role of IP‐10 in autoimmune liver diseases, we studied the serum levels of IP‐10 in 14 patients with autoimmune hepatitis (AIH), 23 patients with primary biliary cirrhosis (PBC), and 65 patients with chronic viral hepatitis (20 type B and 45 type C). The hepatic expression of IP‐10 mRNA and the correlation between the serum levels of IP‐10 and clinical parameters were also evaluated. In addition to 20 healthy controls, 16 rheumatoid arthritis (RA) patients were included as an extrahepatic inflammatory disease. The serum level of IP‐10 was significantly (P < 0·02) higher in patients with AIH, PBC, and chronic hepatitis B and C than in healthy controls, and it was significantly correlated (P < 0·05) with the serum levels of aspartate aminotransferase and alanine aminotransferase in patients with AIH, PBC, and chronic hepatitis B and C. The serum level of IP‐10 was not elevated in RA patients. After successful treatment of AIH and chronic hepatitis C, the serum level of IP‐10 decreased to the same level as in healthy volunteers. As we previously showed in cases with chronic hepatitis B or C, in situ hybridization in both AIH and PBC cases demonstrated the expression of IP‐10 mRNA in hepatocytes around focal or lobular necrosis surrounded by infiltrating mononuclear cells, whereas IP‐10 mRNA was not expressed in areas around the damaged bile ducts in PBC cases. The present results suggest that IP‐10 is specifically produced by hepatocytes in inflammatory areas irrespective of the aetiology of hepatitis, and that IP‐10 may help to recruit T cells to the hepatic lesions in autoimmune liver diseases as well as in chronic viral hepatitis.

Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment

BACKGROUND/AIMS The emergence of lamivudine-resistant hepatitis B virus (HBV) was reported in patients with prolonged lamivudine administration. There was no report of the existence of tyrosine-methionine-aspartate-aspartate (YMDD) mutant in non-lamivudine treated chronic hepatitis B patients. In the present study, we developed a sensitive assay and applied it to the detection of YMDD mutant. METHODS We developed peptide nucleic acid (PNA) mediated polymerase chain reaction clamping for detecting mutations in a YMDD motif of the hepatitis B virus DNA polymerase gene. We studied YMDD mutants in a patient with HBV DNA breakthrough longitudinally and in non-lamivudine treated patients (36 patients). RESULTS We could detect as little as 0.01-0.001% of mutant viruses coexisting in 10(5)-10(9) copies of wild-type viruses using this assay. YMDD mutant was detected 7 months before clinical breakthrough, which was 6 months earlier than using the conventional restriction fragment length polymorphism assay. YMDD mutants were also detected in four of 18 anti-HBe antibody positive untreated chronic hepatitis type B: YMDD+tyrosine-valine-aspartate-aspartate (YVDD) in two patients and YMDD+tyrosine-isoleucine-aspartate-aspartate (YIDD) in two patients, however, none in HBe antigen positive patients. CONCLUSIONS We developed a highly sensitive assay for detecting YMDD mutants. This is an effective procedure for monitoring patients during or before lamivudine treatment and can provide more insights into the therapeutic strategies for chronic hepatitis B patients.

Clinical significance of elevated serum interferon- inducible protein-10 levels in hepatitis C virus carriers with persistently normal serum transaminase levels.

The aim of this study was to assess the immuno‐ logical profile in hepatitis C virus carriers with persistently normal serum transaminase levels. Forty‐two serum HCV RNA positive patients with persistently normal serum transaminase levels (22 natural ‘asymptomatic HCV carriers’ and 20 biochemical responders to IFN therapy) and 23 complete responders to IFN therapy were enrolled. The HCV genotypes and serum HCV RNA levels were determined before IFN therapy in treatment responders, and at entry in the others. The serum levels of IFN‐inducible protein‐10 (IP‐10) (a protein mainly induced by IFN‐γ), interleukin (IL)‐10, and IL‐4 were measured in all patients while the serum transaminase levels were normal. The serum transaminase levels and platelet counts were then monitored for the next 4 years and the changes in liver fibrosis were assessed. The serum levels of IP‐10 in infected and biochemically normal patients were significantly higher than the levels in complete responders to therapy, whereas the serum levels of IL‐10 and IL‐4 did not vary significantly among the different groups. During the 4‐year follow‐up period, 10/20 (50%) biochemical responders and 12/22 (55%) asymptomatic carriers had an elevation of the serum transaminase levels. A significant (P=0.0370) increase in platelet count after 4 years and improvement in liver fibrosis were noted in treatment responders but not in infected patients. The weak but significant residual immune response as reflected by the increased serum IP‐10 level may underlie the outcome of HCV carriers with persistently normal serum transaminase levels.

Involvement of Kupffer cells in the interaction between neutrophils and sinusoidal endothelial cells in rats.

During endotoxic liver injury, large numbers of neutrophils infiltrate the liver, and serum levels of tumor necrosis factor-&agr; (TNF-&agr;) become elevated. The object of this study was to assess the roles of TNF-&agr; secreted by Kupffer cells in the interaction between neutrophils and sinusoidal endothelial cells (SECs). Rat neutrophils were perfused onto SECs that were stimulated with either TNF-&agr; or supernatant from lipopolysaccharide (LPS)-stimulated Kupffer cells using an in vitro flow system. Numbers of adhered or migrated neutrophils were counted, and the effect of an antibody against intercellular adhesion molecule-1 (ICAM-1) was studied. Compared with controls (200 ± 21 cells/mm2), neutrophil adhesion to SECs was significantly increased by both TNF-&agr; (342 ± 26 cells/mm2;P < 0.05) and LPS-stimulated Kupffer cell supernatant (331 ± 29 cells/mm2;P < 0.05). Anti-ICAM-1 significantly inhibited neutrophil adhesion (139 ± 10 cells/mm2;P < 0.05) and decreased the migration rate of neutrophils on SECs treated with LPS-stimulated Kupffer cell supernatant (P < 0.05). LPS-stimulated Kupffer cells secreted TNF-&agr; in an LPS dose-dependent manner, and they significantly enhanced ICAM-1 expression on SECs (P < 0.05 vs. control). In addition, dexamethasone suppressed TNF-&agr; production by LPS-stimulated Kupffer cells and decreased ICAM-1 expression and neutrophil adhesion on SECs. These findings suggest that Kupffer cells are involved in neutrophil adhesion and migration in hepatic sinusoids via TNF-&agr; production and induction of ICAM-1 expression on SECs during liver injury associated with endotoxemia.

Transient biochemical response in interferon therapy decreases the development of hepatocellular carcinoma for five years and improves the long-term survival of chronic hepatitis C patients

The development of hepatocellular carcinoma (HCC) was significantly reduced in both sustained responders (SR) and transient biochemical responders (TR) in chronic hepatitis C (CH-C) patients who received interferon (IFN) therapy. However, the long-term clinical outcome of TR remains unclear. One thousand three hundred and seventy CH-C Japanese patients who received IFN therapy and 54 control cirrhotic patients were enrolled. TR were defined as those patients who showed a normal serum alanine aminotransferase level (<==30 IU/l) at the end of therapy and then relapsed. Mean follow-up period was 5.6 years (6.1 years in 48 cirrhotic patients) in the IFN group and 8.3 years in the 54 control cirrhotic patients. HCC was detected in 114 patients in the IFN group among whom 4 were in the 425 SR, 21 were in the 359 TR and 89 were in the 586 non-responders (NR). The cumulative incidence of HCC was significantly (P=0.0001) inhibited in both SR and TR compared with NR. Its inhibitory effect in TR was within 5 years. Platelet count did not significantly decrease for 2-4 years after IFN therapy in TR, but it significantly decreased in NR 2 years after IFN therapy. The cumulative survival in both SR and TR was significantly higher than NR (SR vs NR; P=0.0001, TR vs NR; P=0.0305). These results indicate that IFN therapy lowers the rate of the progression of HCC and improves the long-term survival even in CH-C patients who transiently respond to IFN therapy.

Clinical characteristics and prevalence of GB virus C, SEN virus, and HFE gene mutation in Japanese patients with nonalcoholic steatohepatitis

BackgroundThis study was carried out to clarify differences in clinical characteristics between fatty liver and nonalcoholic steatohepatitis in a Japanese population, and to assess the significance of GB virus C (GBV-C) infection, SEN virus (SENV) infection, and HFE gene mutation in the pathophysiology of these conditions.MethodsTwenty patients with nonalcoholic steatohepatitis and 18 patients with simple steatosis were enrolled, and their clinical characteristics and histological findings were compared. Detection of GBV-C RNA and SENV DNA was performed by polymerase chain reaction (PCR). Mutational analysis of the HFE gene was performed by PCR-restriction fragment length polymorphism (RFLP).ResultsSerum aspartate aminotransferase (AST) and ferritin were significantly higher (P < 0.05, for both) in NASH than in simple steatosis, and serum total cholesterol (T-Chol) was significantly lower (P < 0.05) in NASH than in simple steatosis. While GBV-C was detectable in the serum of only one patient with NASH, SENV was detected in 50% (15/30) of the patients whose sera were tested for this virus, but the prevalence was not significantly different between the two groups (42% [8/19] in simple steatosis and 64% [7/11] in NASH). The sex ratio, body mass index (BMI), and age were not significantly different between the two groups, and mutation in the HFE gene was not detected in any patient.ConclusionsHigher serum AST and ferritin, and lower serum T-Chol are distinctive features in NASH when compared with simple steatosis. GBV-C infection, SENV infection, and HFE gene mutation were not considered to influence the development of NASH from simple fatty liver.

Activated Kupffer cells play an important role in intra-hepatic Th1-associated necro-inflammation in Concanavalin A-induced hepatic injury in mice

BACKGROUND/AIMS: To examine whether or not activated Kupffer cells play an important role in intra-hepatic Th1-associated necro-inflammation in Concanavalin A (Con A)-induced hepatic injury in mice. METHODS: Con A was administered to Balb/c mice pretreated with or without gadolinium chloride (GdCl(3)). Kupffer cell activation was evaluated by their ability to produce superoxide anions in situ under liver perfusion with nitro blue tetrazolium (NBT). Hepatic concentration of cytokines was measured by ELISA and the mRNA expression of CXC chemokine receptor 3 (CXCR3) was evaluated by RT-PCR. Immunohistochemical detection of CD4 positive lymphocytes in the liver was also performed. RESULTS: GdCl(3)-pretreatment significantly (P<0.01) reduced the serum levels of alanine aminotransferase (ALT) in Con A-treated mice. Formazan deposition in Kupffer cells, the hepatic concentration of tumor necrosis factor-alpha and interferon-gamma, the mRNA expression of CXCR3 and the CD4 positive lymphocytes in the liver were decreased in GdCl(3)-pretreated mice as compared with those without GdCl(3)-pretreatment (P<0.05, respectively). CONCLUSIONS: Activated Kupffer cells, which produce superoxide anions, are involved in Con A-induced hepatic necro-inflammation in mice possibly through the activation of Th1-associated immune response mediated by CD4 and/or CXCR3 positive cells recruited into the liver.

Non‐prescription supplement‐induced hepatitis with hyperferritinemia and mutation (H63D) in the HFE gene

A 55‐year‐old Japanese woman was hospitalized because liver function tests showed an abnormality. Transaminases and biliary enzymes were markedly elevated with hyperferritinemia. Her imaging tests revealed no significant abnormality. She had been taking various non‐prescription supplements for over approximately 6 months. After the supplements were discontinued her liver function gradually improved. This clinical course was suggestive of supplement‐induced hepatitis. She had no history of taking supplements containing iron, so it was interesting that she had hyperferritinemia. We examined C282Y and H63D, which are important mutations in theiron‐metabolizing gene, HFE. She was found to be heterozygous for the H63D mutation. The interaction between hyperferritinemia and supplements is unknown, but it can be speculated that some interaction between iron overload and supplements may have underlain the pathogenesis of her liver injury.

Efficacy of daclatasvir plus asunaprevir in patients with hepatitis C virus infection undergoing and not undergoing hemodialysis: Daclatasvir plus asunaprevir in non-HD and HD patients

To evaluate the virologic responses and clinical course of daclatasvir plus asunaprevir treatment in non‐hemodialysis (non‐HD) and hemodialysis (HD) patients infected with genotype 1 hepatitis C virus (HCV).

Examination time as a quality indicator of screening upper gastrointestinal endoscopy for asymptomatic examinees

The significance of examination time of esophagogastroduodenoscopy (EGD) for asymptomatic examinees is yet to be established. We aimed to clarify whether endoscopists who allot more examination time can detect higher numbers of neoplastic lesions among asymptomatic examinees.