Hiroyuki Saito

Immunohistochemical detection of 4-hydroxy-2-nonenal-modified-protein adducts in human alcoholic liver diseases

4-Hydroxy-2-nonenal (HNE) is one of the major components of lipid peroxidation product and has been shown to react with proteins to form HNE-protein adducts. HNE-protein adducts are relatively stable and can be used as a marker of radical-mediated cellular damage. We report herein the immunohistochemical analysis of HNE-protein adducts in human alcoholic liver diseases using a specific monoclonal antibody HNEJ-2. Cytoplasm of hepatocytes and bile duct epithelia was positively stained for HNE-protein adducts, and the nucleus was negligibly stained. The immunohistochemical intensity of hepatocytes was classified into three groups: strong, moderate, and faint staining. Strong staining was found in 43% of alcoholic liver diseases and in 4% of viral liver diseases. Hepatocytes of alcoholic liver diseases contained a higher amount of HNE-protein adducts than those of viral liver diseases, and the difference was statistically significant (p = 0.005; chi2 test). Semiquantitative analysis of the histological intensities of HNE-protein adducts and iron indicated a significant positive correlation (p = 0.084; Spearmans rank correlation). The localization of HNE-protein adducts and iron in hepatocytes appeared to be identical. These data suggested the correlation between HNE-protein adducts and iron. Our results indicate that HNE-protein adducts, a marker of oxidative stress-induced damage, are increased in human alcoholic liver damage, and that hepatic siderosis may act on the production of free radicals.

Significant increase of interleukin 6 production in blood mononuclear leukocytes obtained from patients with active imflammatory bowel disease

In the present study, we compared the potency of interleukin 6 production in peripheral blood mononuclear leukocytes between paired patients with active stage and inactive stage of inflammatory bowel disease. Subjects included nine patients with ulcerative colitis, ten patients with Crohns disease and sex-matched nine healthy volunteers. Mononuclear leukocytes were stimulated with concanavalin A for 24 h to induce interleukin 6 production. Interleukin 6 content in the culture medium was assayed by using specific ELISA and interleukin 6 dependent cell line MH-60. Interleukin 6 production was found to be significantly increased in mononuclear leukocytes from both active ulcerative colitis and Crohns disease as compared to that from control subjects. There was no significant difference in interleukin 6 production between ulcerative colitis and Crohns disease. The potency of interleukin 6 production was returned to the control level when the diseases became inactive. The present results, therefore, may indicate some important role of interleukin 6 in the pathogenesis of inflammatory bowel disease and also the potency of interleukin 6 production in mononuclear leukocytes can be an indicator of the activity of inflammatory bowel disease.

Evaluation of Influenza Virus A/H3N2 and B Vaccines on the Basis of Cross-Reactivity of Postvaccination Human Serum Antibodies against Influenza Viruses A/H3N2 and B Isolated in MDCK Cells and Embryonated Hen Eggs

ABSTRACT The vaccine strains against influenza virus A/H3N2 for the 2010-2011 season and influenza virus B for the 2009-2010 and 2010-2011 seasons in Japan are a high-growth reassortant A/Victoria/210/2009 (X-187) strain and an egg-adapted B/Brisbane/60/2008 (Victoria lineage) strain, respectively. Hemagglutination inhibition (HI) tests with postinfection ferret antisera indicated that the antisera raised against the X-187 and egg-adapted B/Brisbane/60/2008 vaccine production strains poorly inhibited recent epidemic isolates of MDCK-grown A/H3N2 and B/Victoria lineage viruses, respectively. The low reactivity of the ferret antisera may be attributable to changes in the hemagglutinin (HA) protein of production strains during egg adaptation. To evaluate the efficacy of A/H3N2 and B vaccines, the cross-reactivities of postvaccination human serum antibodies against A/H3N2 and B/Victoria lineage epidemic isolates were assessed by a comparison of the geometric mean titers (GMTs) of HI and neutralization (NT) tests. Serum antibodies elicited by the X-187 vaccine had low cross-reactivity to both MDCK- and egg-grown A/H3N2 isolates by HI test and narrow cross-reactivity by NT test in all age groups. On the other hand, the GMTs to B viruses detected by HI test were below the marginal level, so the cross-reactivity was assessed by NT test. The serum neutralizing antibodies elicited by the B/Brisbane/60/2008 vaccine reacted well with egg-grown B viruses but exhibited remarkably low reactivity to MDCK-grown B viruses. The results of these human serological studies suggest that the influenza A/H3N2 vaccine for the 2010-2011 season and B vaccine for the 2009-2010 and 2010-2011 seasons may possess insufficient efficacy and low efficacy, respectively.

Induction of transferrin receptor by Ethanol in Rat primary hepatocyte culture

BACKGROUNDnIt is not uncommon for alcoholics to have iron accumulation in the liver, a condition that may contribute to the development of alcoholic liver disease. Recently, we reported that the expression of transferrin receptor, which mediates cellular iron uptake, was increased in hepatocytes in patients with alcoholic liver disease. To elucidate the mechanism of the iron accumulation in hepatocytes in such disease, we examined whether ethanol exposure induced the transferrin receptor expression and increased the cellular iron uptake.nnnMETHODSnRat primary hepatocytes were isolated and cultured in the presence of 20 micromol/liter of iron and 25 mmol/liter of ethanol.nnnRESULTSnEthanol exposure to the hepatocytes demonstrated an ~2-fold increase in transferrin receptor expression for 24 hr, shown by Western blot analysis and S-methionine metabolic labeling, 19% increase in Fe-transferrin uptake by hepatocytes, and 20% increase in activity of iron regulatory protein examined by band shift assay.nnnCONCLUSIONnEthanol exposure induced the transferrin receptor expression, partially through the activation of iron regulatory protein, and increased the transferrin-bound iron uptake in rat hepatocyte cultures. The induction of transferrin receptor by ethanol might be one of the mechanisms of iron accumulation in the hepatocytes in alcoholic liver disease.