Katsumi Yamauchi

Significance of Fas antigen-mediated apoptosis in human fulminant hepatic failure

Abstract OBJECTIVE: The aim of this study was to elucidate the role of apoptosis in human fulminant hepatic failure. We studied the expression of Fas antigen on liver tissues, Fas ligand in lymphocytes, and soluble Fas ligand in patients’ serum. METHODS: On finding apoptotic cells in fulminant hepatic failure liver, we first examined them using the TUNEL method. Subsequently, the expression of Fas was studied by immunostaining. Simultaneously, Fas ligand presenting on both liver-infiltrated cells and peripheral lymphocytes was studied by reverse transcription-polymerase chain reaction, and soluble Fas ligand in sera was measured by ELISA. RESULTS: By using the TUNEL method, we first demonstrated that many apoptotic cells existed in fulminant hepatic failure but not in normal ones. Our immunohistochemistry study showed that many hepatocytes in fulminant hepatic failure strongly expressed Fas. In addition, Fas ligand on both liver-infiltrating lymphocytes and peripheral lymphocytes in fulminant hepatic failure patients was detected. The serum level of soluble Fas ligand was significantly increased in fulminant hepatic failure (mean value, 2.91 ng/ml in fulminant hepatic failure [n = 10], 1.62 ng/ml in acute hepatitis [n = 10], and 0.27 ng/ml in healthy controls [n = 10]). Furthermore, this serum level of sFas ligand was significantly associated with prothrombin time both in acute hepatitis and fulminant hepatic failure. CONCLUSIONS: The present results indicate that Fas-mediated apoptosis may be one of the triggers for the induction of fulminant hepatic failure.

A molecular analysis of viral persistence in surface antigen-negative chronic hepatitis B.

To identify the mechanisms of viral persistence in patients with chronic hepatitis B after the acquisition of anti‐hepatitis B surface antigen antibodies (antiHBs), we serially analyzed the nucleotide sequence of the envelope region in a cohort of infected patients. Four patients with histological diagnoses of chronic hepatitis B who had at least 5 years of observance by our hospital staff were studied. All but one showed normalization of serum alanine aminotransferase (ALT) concentration after clearance of the hepatitis B surface of antigen (HBsAg) and the appearance of anti‐HBs. Hepatitis B virus (HBV) DNA was still detectable by polymerase chain reaction (PCR) amplification assay in serum specimens from two patients, even in the presence of circulating anti‐HBs. The envelope gene was amplified by PCR in serum samples obtained both before and after seroconversion, and direct cycle sequencing of the PCR products was performed. A mutation resulting in a premature stop codon was found in the pre‐S1 region of one patient just prior to clearance of HBsAg. Two years later, the stop codon was converted to a leucine codon and three mutations developed in the “a” loop. In the other patient, 16 amino acids had been deleted between amino acids 8 and 23 in the pre‐S2 region before clearance of HBsAg. After the appearance of circulating anti‐HBs, the pre‐S2 gene reverted to the wild type but three additional mutations appeared inside the “a” loop. These results suggest that HBV mutates when HBsAg is cleared, which may contribute to viral persistence due to an evasion of the host immune surveillance.

Significance of Soluble TNF Receptor-I in Acute-Type Fulminant Hepatitis

OBJECTIVE:Fulminant hepatitis is associated with apoptosis of hepatocytes, which is mediated via Fas and tumor necrosis factor (TNF) receptors. The clinical significance of apoptotic factors and these receptors was investigated in fulminant hepatitis.METHODS:Serum levels of TNF-α, soluble TNF receptor-I and -II, soluble Fas antigen, and Fas ligand were measured. Then, the relationships between these parameters and the severity or prognosis of fulminant hepatitis were studied.RESULTS:Serum levels of TNF-α, soluble TNF receptor-I, and soluble TNF receptor-II were increased in acute-type fulminant hepatitis. In particular, soluble TNF receptor-I was significantly higher than in patients with subacute-type fulminant hepatitis, severe acute hepatitis, acute hepatitis, or healthy controls. The soluble TNF receptor-I level continued to increase or remained high in patients who died of acute-type fulminant hepatitis, and eight of nine patients had a level >10 ng/ml. In contrast, the soluble TNF receptor-I level remained <10 ng/ml in survivors. Soluble Fas and soluble Fas ligand levels tended to increase in all types of acute liver disease and were not specific to fulminant hepatitis.CONCLUSION:Our findings suggested that monitoring the soluble TNF receptor-I level may help to assess the prognosis of acute-type fulminant hepatitis and that TNF might be associated with massive hepatic necrosis.

Elevated serum interleukin-6 levels in patients with acute hepatitis

To study the mechanisms of hepatocyte injury, we examined serum interleukin-6 (IL-6) level in acute hepatitis patients. Based on their clinical features, these patients were divided into three groups, acute hepatitis (AH), severe acute hepatitis, and fulminant hepatic failure (FHF). The present study demonstrated that, in association with their clinical status, their serum IL-6 levels were gradually increased (16.5±14.5 pg/ml in AH, 26.3±19.0 pg/ml in severe AH, and 470.2±261.4 pg/ml in FHF; control level, 5.2±0.6 pg/ml). Furthermore, we found that a significant correlation between serum IL-6 level and prothrombin time existed in these patients and that the elevated serum IL-6 returned to a normal range after recovery from their hepatocyte injury. Thus, our study demonstrates that the serum IL-6 level is a possible marker for identifying the clinical status in acute hepatitis and that this cytokine may have some roles in hepatocyte injury.

Influence of TNF gene polymorphism in patients with acute and fulminant hepatitis

BackgroundTumor necrosis factor (TNF) is involved in liver damage, especially in fulminant hepatitis (FH). Our previous data showed that the serum level of TNF-α was markedly increased in FH. To investigate the mechanism of the overproduction of TNF in FH patients, polymorphism of the TNF gene was studied.MethodsWe analyzed 120 healthy subjects (controls), 63 patients with acute hepatitis (AH), and 32 patients with FH. Of the 32 FH patients, 21 died or received liver transplantation (FH-D), and 11 survived with intensive therapy (FH-S). The TNF-α promoter region at −1031, −863, −857, −308, and −238, and TNF-β Nco1 polymorphism sites were studied.Results(1) The four groups showed no differences in polymorphisms of positions −857, −308, and −238. The allelic frequencies of positions −1031C and −863A in the FH-D patients were significantly higher compared to findings in control subjects. (2) The allelic frequency of B2 in the TNF-β gene was significantly higher in FH patients, and particularly in the FH-D patients, compared to control subjects. (3) When the patients were divided into four groups by etiology, hepatitis A virus (HAV), HBV, HCV, and non-A non-B non-C, the allelic frequencies of positions −863A and TNF-β B2 in FH patients were increased in the non-A non-B non-C group compared to controls.ConclusionsFH patients with a poor prognosis had higher frequencies of positions −1031C and −863A in the TNF-α promoter region, and higher frequencies of the B2 allele of the TNF-β gene. These data suggest that the genomic background may be associated with the prognosis of acute liver failure.

Analytical study of the clinical response to two distinct adoptive immunotherapies for advanced hepatocellular carcinoma: comparison between LAK cell and CTL therapy.

To evaluate the effect of two distinct adoptive immunotherapies, tumor-specific cytotoxic T-cell (CTL) therapy and lymphokine-activated killer (LAK) cell therapy, the clinical responses of patients with stage IV primary hepatocellular carcinoma (HCC) treated with these therapies were studied. Of 18 patients treated with CTL, 3 had complete regression (CR), 2 had partial regression, and 3 had minor regression (MR). Their median survival was 21 months after the end of therapy, and 1 CR patient survived for > 6 years. On the other hand, in the LAK-cell-treated group of eight patients, four had MR and their median survival was only 2 months. No survival was observed 27 months after the end of LAK cell therapy. These results indicate that tumor-specific CTL therapy is more effective than LAK cell therapy and that it might be a promising therapeutic tool for advanced HCC patients.

Analysis of adhesion molecules in patients with idiopathic portal hypertension

Background: The aetiology of idiopathic portal hypertension (IPH) is unknown. However, some evidence of immunological abnormalities in IPH patients has been reported.

Predominant T helper 1 cells in patients with idiopathic portal hypertension.

Background: The pathologic mechanism of idiopathic portal hypertension (IPH) is unknown. Because cytokines and the balance of T helper (h) 1 and Th2 CD4+ T cells have been reported to be important for regulating the immune response, in the present study we investigated the role of cytokines and the distribution of cytokine‐producing cells in IPH patients.

Clinical significance of autoantibody to hepatocyte membrane antigen in type 1 autoimmune hepatitis

OBJECTIVE:By using HepG2 as flow cytometry target, we have reported that autoantibody to hepatocyte membrane antigen (anti-HMA) was frequently found in autoimmune hepatitis (AIH) patients. In this study, we have examined this autoantibody in relation to clinical features in these patients.METHODS:HepG2 cells were incubated with diluted serum and subsequently with FITC-conjugated antihuman immunoglobulin. The results were expressed as relative fluorescence intensity. The prevalence of anti-HMA was estimated by setting the upper limit of mean ± 3 SD obtained from healthy subjects.RESULTS:We found that the mean relative fluorescence intensity was 1.67 ± 0.5 in AIH with low serum ALT level (group 1 AIH), 4.20 ± 1.9 in AIH with high serum ALT level (group 2 AIH), and 1.92 ± 0.9 in age-matched chronic hepatitis C virus-positive patients. Their positive rate was 37.5% (three of eight) in group 1 AIH, 95.0% (19 of 20) in group 2 AIH, and 33.3% (four of 12) in chronic hepatitis C patients. In 12 group 2 AIH patients, their mean relative fluorescence intensity was significantly decreased during immunosuppressive therapy. The association between serum ALT level and anti-HMA was confirmed by the facts that a significant direct quantitative relationship existed between these two levels and by serial studies of anti-HMA in four AIH patients. Anti-HMA was also detected in five non-B, non-C hepatitis patients having clinical features resembling those of AIH.CONCLUSIONS:The present results have shown that the anti-HMA was tightly associated with the degree of hepatocyte inflammation and that the measurement of anti-HMA may have some advantage in clinical evaluation of some of non-B, non-C hepatitis patients.

Influence of TNF gene polymorphism and HLA-DRB1 haplotype in Japanese patients with chronic liver disease caused by HCV

OBJECTIVE:To clarify the host genomic role in chronic liver disease associated with hepatitis C virus (HCV), we investigated the relationship between the severity of hepatitis and the polymorphisms of the tumor necrosis factor (TNF) gene or the human leukocyte antigen (HLA)-DRB1 haplotypes.METHODS:We analyzed 40 healthy subjects, 50 patients with chronic inactive hepatitis caused by HCV with mean serum ALT concentrations under 40 IU/ml (group A), and 50 patients with chronic active liver disease caused by HCV and mean ALT concentrations over 50 IU/ml (group B).RESULTS:There were no significant differences in the frequencies of TNF promoter gene variants at positions −238 and −308 between the groups. Regarding polymorphisms at the TNF-β NcoI site, the frequency of B1B1 homozygotes in group A was significantly increased, compared with the healthy subjects and those in group B (controls 7.5%, group A 34%, group B 10%). Regarding the analysis of HLA-DRB1, DRB1*0901 was significantly more frequent in group A than in group B (group A 19%, group B 5%). TNF B1B1 homozygotes were associated with HLA-DRB1*0901 and *1302, and negatively associated with DRB1*0405. Combination analysis revealed that HCV was inactive in the majority of patients who were both DRB1*0901 and B1B1 homozygotes.CONCLUSION:Our data suggest that TNF gene polymorphisms and HLA-DRB1 haplotype may influence the activity of HCV in chronic liver disease.