Adrian L. W. F. Eddleston

Antibodies to the Surface of Halothane-Altered Rabbit Hepatocytes in Patients with Severe Halothane-Associated Hepatitis

Circulating antibodies reacting specifically with the cell membrane of hepatocytes isolated from halothane-anesthetized rabbits were detected in nine of 11 patients with fulminant hepatic failure after helothane-induced anesthesia. The immunoglobulin deposition, as revealed by immunofluorescence, showed a granular pattern on the hepatocyte surface membrane. Preincubation of halothane-pretreated, but not of control, hepatocytes with serum containing this antibody rendered them susceptible to cytotoxic effects of normal lymphocytes in vitro. Control studies using serum from subjects repeatedly exposed to halothane without the development of liver damage, and from patients with viral and toxic liver injury have confirmed the specificity of these findings to serve halothane-associated liver injury. These results provide further evidence of an immunologic component in this condition.

Increased production of tumour necrosis factor alpha in chronic hepatitis B virus infection

Plasma tumour necrosis factor alpha (TNF) and in-vitro TNF production by peripheral blood mononuclear cells (PBMC) were studied in 22 HBsAg seropositive patients and compared with 23 normal and 10 disease controls. Plasma TNF and unstimulated TNF production correlated (Rs = 0.55, p = 0.012) and were significantly elevated in HBsAg and HBeAg seropositive patients (p less than 0.001, p = 0.006) compared with normal controls. TNF production was also elevated in these patients when PBMC were stimulated with interferon-gamma (p less than 0.05) or LPS (p = 0.035). Plasma TNF and TNF production in HBsAg anti-HBe seropositive subjects were not elevated. TNF production in unstimulated cells correlated with serum HBV DNA level (R = 0.53, p = 0.02) but not with serum aspartate transaminase (AST) or histological activity. It is concluded that PBMC are activated to produce TNF both spontaneously and in response to second stimuli in chronic hepatitis B virus infection and that this activity is related to the presence of viral replication.

Hepatocellular carcinoma in Great Britain: influence of age, sex, HBsAg status, and aetiology of underlying cirrhosis.

An analysis of 294 patients who died with cirrhosis showed that 24% had developed hepatocellular carcinoma. Haemochromatosis and HBsAg positive chronic active hepatitis were high risk groups (36% and 42% respectively) and the frequency was lowest in primary biliary cirrhosis and HBsAg negative chronic active hepatitis (3% and 11% respectively). Those with hepatocellular carcinoma showed a striking male preponderance (11:1) and further analysis has shown that the proportion developing this tumour in each group was closely related to the proportion of males in that group (r=0.97). Age was the only other significant factor, malignant change occurring more commonly in those over the age of 50 years than those below (30% and 7% respectively, P less than 0.005). The indluence of HBsAg was largely accounted for by the known predisposition of males to carry HBsAg. The group of patients who had developed this tumour without cirrhosis were younger (mean age 39 years) and had a lower male to female ratio of 1.1:1 and the place of contraceptive-related tumour within this group is dicussed.

Cell-mediated immunity and suppressor-T-cell defects to liver-derived antigens in families of patients with autoimmune chronic active hepatitis.

By means of an indirect T-lymphocyte migration inhibitory factor assay, T-cell sensitisation to the asialoglycoprotein receptor, a liver-specific protein on the surface of hepatocytes, was found in patients with autoimmune chronic active hepatitis (CAH) but not in healthy relatives or spouses. Cellular immunity to a liver-derived lipoprotein complex (LSP) which also contains the asialoglycoprotein receptor, and to which immune reactions have been previously demonstrated in patients with CAH, was also examined. In contrast to the findings with the purified asialoglycoprotein receptor, cell-mediated sensitisation to LSP was detected in all the patients and also in 24% of first-degree relatives and 27% of spouses, suggesting an environmental influence. There was a functional defect in suppressor T lymphocytes specific for liver-derived antigens in 50% of first-degree relatives and 43% of second-degree relatives, but in only 9% of spouses. This abnormality of immunoregulation may be genetically determined and crucial to the development of the disease.

Ethanol metabolism in the generation of new antigenic determinants on liver cells.

Antibodies directed against ethanol altered liver cell components have been detected in the serum of nearly 50% of patients with alcoholic liver disease although the pathogenetic mechanisms are unclear. The importance of ethanol metabolism in the generation of new antigenic determinants on liver cells was investigated by in vivo inhibition of alcohol or acetaldehyde dehydrogenase and an induced cytotoxicity assay. There was a significant reduction in cytotoxicity to hepatocytes isolated from rabbits treated with ethanol 1 g/kg when the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was inhibited. In contrast when the oxidation of acetaldehyde was inhibited by disulfiram cytotoxicity was significantly enhanced. These results show that ethanol metabolism is integral to the expression of the ethanol related determinant and suggest that an impaired ability to metabolism acetaldehyde could lead to the development of immunological reactions to alcohol altered liver membrane antigens.

Polymorphism in the Human Complement C4 Genes and Genetic Susceptibility to Autoimmune Hepatitis

Susceptibility to autoimmune hepatitis is associated with the HLA-DR3 and DR4 haplotypes, but which genes are directly involved in the pathogenesis, has not been established. Low levels of complement component C4 and elevated frequencies of C4 null allotypes have been described in patients, suggesting that the C4 genes, which are closely linked with the HLA loci, may play a role. We therefore examined restriction fragment length polymorphisms in the C4 and 21-hydroxylase genes, and determined HLA-A and B phenotypes, and HLA-DR, DQ and DP genotypes in a large series of Caucasoid patients with autoimmune hepatitis and matched controls. A DNA deletion of the C4A gene and the 21-hydroxylase A pseudogene was found to be present in 50% of patients compared to 23% of controls (Pc < 0.005, relative risk = 3.3). This increase, however, appears to be due to linkage disequilibrium with HLA-DR52a which was most strongly associated with the disease. Complete C4A deficiency, determined by homozygosity for the deletion increased the risk to 18.1 (16% versus 1%, Pc < 0.005), suggesting an additional role for C4 in disease susceptibility. C4 deletions were associated with an increased mortality and tendency to relapse whilst on treatment but did not correlate with age of onset of disease. Our data suggest that MHC-encoded susceptibility to autoimmune hepatitis is polygenic, involving the HLA-DR genes plus other loci, and C4 deficiency may be a marker of disease susceptibility and/or severity.

Autoimmunity to a liver membrane lipoprotein and liver damage in alcoholic liver disease.

Antibodies reacting with a liver membrane lipoprotein (LSP) have been detected by radioimmunoassay in the sera of 15 (27%) of 55 patients with alcohol-related liver lesions. There was a close association between the presence of the anti-LSP antibody and the findings on liver biopsy of a lymphocytic infiltrate in the portal tracts together with piecemeal necrosis of periportal hepatocytes. These histological features are characteristically found in the autoimmune disorder of chronic active hepatitis, in which anti-LSP antibodies are almost invariably present. It is suggested that in these cases of alcoholic liver disease there is loss of tolerance, and continued production of anti-LSP could promote periportal inflammation and accelerate the progression to cirrhosis. In the cases of acute alcoholic hepatitis without periportal inflammation studied, anti-LSP was not detected demonstrating that production of this autoantibody is not simply secondary to liver damage.

Serological markers in fulminant hepatitis B.

Serological markers for hepatitis B virus infection have been examined in 34 patients with acute hepatitis B, 17 of whom developed fulminant hepatic failure. Hepatitis B surface antigen concentrations were significantly lower and hepatitis Be antigen was less frequently detectable in patients with fulminant hepatic failure compared with those with acute hepatitis (median 0.64 micrograms, range 16-0 and median 32 micrograms and range 100-4 micrograms respectively, p less than 0.001; HBeAg detected in 12% and 88% respectively, p less than 0.001). The IgM component of hepatitis B core antibody was significantly higher in the patients with fulminant hepatic failure with median values of 500 IU/ml compared with those with uncomplicated hepatitis (median 202 IU/ml, p less than 0.05 Wilcoxons rank test). Three patients who developed a fulminant course had detectable levels of either anti-HBs or anti-HBe. These results are consistent with enhanced antibody responses to all three hepatitis B virus antigens and more rapid clearance of the latter during fulminant hepatic failure.

The major histocompatibility complex influences the development of chronic liver disease in male children and young adults with cystic fibrosis

BACKGROUND/AIMS Chronic liver disease is a well-recognised complication of cystic fibrosis. Recent reports suggest that its development is not determined by specific mutations within the cystic fibrosis gene; however, familial clustering of portal hypertension cases and inappropriate immune responses against liver membrane antigens demonstrated in children with cystic fibrosis and chronic liver disease suggest that other genetic loci may be relevant. As the major histocompatibility complex has an important immunoregulatory role, we have investigated for associations with this complex and chronic liver disease in cystic fibrosis. METHODS We have determined human leucocyte antigen class I (A and B) and class II (DR) phenotypes by serological tissue typing and class II (DR and DQ) and class III (complement component C4 and 21-hydroxylase) gene polymorphisms in 274 children and young adults with cystic fibrosis, of whom 82 had evidence of chronic liver disease with portal hypertension in 49, and 146 healthy controls. RESULTS A marked difference in human leucocyte antigen frequency was limited to DQ6, which was found in 66.7% of cystic fibrosis patients with liver disease compared to 32.9% of patients with no liver disease (Pc < 0.03) and 28.8% of controls (Pc < 0.006). An increased frequency of the two antigens in strong linkage disequilibrium with DQ6 was also observed within this patient group, namely DR15 and B7. When the patients were stratified for the presence of portal hypertension, these observations were confirmed, but the human leucocyte antigen associations were significant only for male patients and there was no association with the age of onset of liver disease. CONCLUSIONS These data suggest that the haplotype B7-DR15-DQ6 may carry an increased risk of development of liver disease in male cystic fibrosis patients.

Detection of hepatitis B virus antigens in liver tissue

Abstract The expression of hepatitis B virus antigens was studied by double staining liver tissue with appropriate antisera and correlated with serum hepatitis B viral DNA and histology in 28 patients with disease related to chronic hepatitis B virus infection. The cellular localization of hepatitis B core and hepatitis B e antigens generally coincided, but there were important differences at a subcellular level. Thus, hepatitis B e antigen was detected in nuclei and/or cytoplasm but strong cytoplasmic hepatitis B e antigen was associated with a high serum hepatitis B viral DNA ( P = 0.0017) but not with active liver disease. Hepatitis B core antigen could also be detected in nuclei and/or cytoplasm, but strong cytoplasmic hepatitis B core antigen expression, exceeding that of hepatitis B e antigen, was associated with active liver disease ( P = 0.041) and not with serum hepatitis B virus DNA. The proportion of hepatocytes expressing hepatitis B surface antigen correlated inversely with the serum titer ( P = 0.0017), whereas hepatitis B surface and nucleocapsid antigens were usually expressed independently. The data support the hypothesis that cytoplasmic hepatitis B core antigen and not hepatitis B e antigen is the target for immune system-mediated cytolysis of hepatocytes. Cytoplasmic hepatitis B e antigen is not associated with liver damage but is instead associated with high levels of hepatitis B virus replication.