Barbara M. McFarlane

Antibodies to hepatitis C virus in autoimmune liver disease: evidence for geographical heterogeneity

To resolve conflicting reports about the occurrence of antibodies against hepatitis C virus (HCV) in patients with autoimmune chronic active hepatitis (AI-CAH), sera from UK and Italian patients were tested with the original anti-HCV assay (Ortho) and a novel anti-HCV assay (UBI) based entirely on synthetic HCV peptides. 28 (60%) of 47 Italian patients with type-1 AI-CAH were anti-HCV-positive by Ortho ELISA, 25 of whom were also strongly positive by the UBI assay. 15 (60%) of 25 UK patients with type-1 AI-CAH were HCV-positive by Ortho ELISA but only 2 were positive by the UBI assay. Similarly, 29 (88%) of 33 Italian patients with type-2 AI-CAH, but 0 of 10 UK patients, were very strongly anti-HCV-positive with the UBI assay. Italian patients with AI-CAH appear to have a high frequency of genuine exposure to HCV, whereas seropositivity by the Ortho HCV ELISA in UK patients is likely to represent a false-positive result. These findings indicate important geographical and/or genetic influences in autoimmune liver disease among different populations.

Serum autoantibodies reacting with the hepatic asialoglycoprotein receptor protein (hepatic lectin) in acute and chronic liver disorders

Circulating autoantibodies reacting with affinity-purified, hepatic asialoglycoprotein receptor protein, hepatic lectin (HL), were detected by radioimmunoassay in 15 (83%) of 18 patients with autoimmune chronic active hepatitis (AI-CAH) who had active disease, at titres that showed a positive correlation (P less than 0.05) with severity of periportal inflammation assessed histologically. In contrast, 10 AI-CAH patients whose disease was in remission were all anti-HL seronegative. Anti-HL was also detected in 16 (73%) of 22 patients with hepatitis B virus-related CAH-a similar frequency to that in active AI-CAH but at significantly lower (P less than 0.005) titres. Only 1 of 8 patients with chronic active liver disease due to presumed non-A, non-B (NANB) viral infection and 5 (22%) of 23 with primary biliary cirrhosis were anti-HL seropositive (P less than 0.001 vs active AI-CAH and HBV-CAH) and there was no correlation with severity of periportal inflammation. Anti-HL antibodies were also found in sera from 7 (35%) of 20 patients with acute virus B hepatitis (AVH-B) but were not detected in 10 patients with AVH-A nor in 12 with AVH due to presumed NANB infection. Anti-HL was not found in sera of 12 patients with autoimmune thyroid disease. Hepatic lectin, a highly purifiable, liver-specific cell surface component, by analogy with the acetylcholine and thyrotropin receptors which are, respectively, targets of the pathogenetically-related autoimmune reactions in myasthenia gravis and autoimmune thyroid disease, may be an important target of autoreactions in liver disease.

Demographics of anti-asialoglycoprotein receptor autoantibodies in autoimmune hepatitis

BACKGROUND/AIMS The asialoglycoprotein receptor (ASGPR) is an established, liver-specific autoantigen. This multicenter study investigated the specificity of anti-ASGPR autoantibodies for autoimmune hepatitis (AIH) in different ethnic groups. METHODS Nine hundred fourteen sera from European, Japanese, and North American (U.S.) patients with chronic inflammatory liver disorders were tested. An enzyme-immunoassay using human ASGPR and a radioimmunoassay against rabbit ASGPR, performed independently on coded sera, were compared. RESULTS The highest frequency (76%) of anti-human ASGPR was found in AIH patients (11/24 U.S.; 21/25 European; 28/30 Japanese), particularly in those with active disease before treatment (53/62, 85%), and decreased in titer with response to immunosuppressive therapy. These antibodies were found at low titers in 43 (11%) of 385 patients with viral hepatitis and in 25 (7.6%) of 328 patients with other chronic inflammatory liver disorders (P < 0.0005 compared with all AIH patients). Twenty of 37 sera tested by enzyme-immunoassay and radioimmunoassay were positive, and nine were negative for anti-ASGPR by both assays (78% concordance); six sera were exclusively positive on human substrate. CONCLUSIONS Circulating anti-ASGPR autoantibodies are closely associated with autoimmune hepatitis independent of geographic or ethnic criteria. Two anti-ASGPR assays currently in use show high reliability.

T-cell-directed hepatocyte damage in autoimmune chronic active hepatitis.

To investigate the function of activated T lymphocytes in autoimmune chronic active hepatitis, 7 of 15 T-cell clones from the peripheral blood of 8 patients were studied. These clones showed specificity for liver-membrane antigen with proliferation when stimulated by rabbit liver cell membranes. 6 of these clones reacted with liver-specific lipoprotein complex, and 1 clone (and 3 subclones) responded to the asialoglycoprotein receptor (ASGPR), both known targets of immune attack in autoimmune chronic active hepatitis. 2 of these clones stimulated autologous B lymphocytes to produce liver-membrane-specific autoantibodies and antibody to the ASGPR. These results suggest that liver-membrane-specific activated T lymphocytes in peripheral blood may be important in the autoimmune attack of chronic active hepatitis.

Organ and Non-organ Specific Autoantibody Titres and IgG Levels as Markers of Disease Activity: A Longitudinal Study in Childhood Autoimmune Liver Disease

No longitudinal study has investigated whether autoantibody titres and serum IgG levels correlate with disease activity in autoimmune liver disease. To determine this, we investigated prospectively 19 patients on 254 occasions between 10 months to 5 years from diagnosis. Nine had anti-nuclear and/or anti-smooth muscle antibody (ANA/SMA) positive autoimmune hepatitis (type 1 AIH), 5 liver kidney microsomal type 1 (LKM-1) positive AIH (type 2 AIH) and 5 ANA/SMA positive autoimmune sclerosing cholangitis (ASC). Correlation between IgG levels, titres of ANA, SMA and LKM-1 and levels of the organ specific autoantibodies anti-liver specific protein (anti-LSP), and anti-asialoglycoprotein receptor (anti-ASGPR) with biochemical evidence of disease activity, as measured by serum aspartate amino transferase (AST) levels, was sought during the course of the disease. AST levels correlated with levels of anti-LSP, anti-ASGPR and IgG in type 1 and 2 AIH, but not in ASC. Positive correlation with AST was also observed for LKM-1 titres in type 2 AIH and for SMA titres in type 1 AIH, but not in ASC. In both AIH and ASC, AST levels correlated with the T cell-dependent immune responses anti rubella IgG and anti tetanus toxoid IgG, but not with the T cell-independent IgG 2 response to pneumococcal capsular polysaccaride. Our results indicate that measurement of organ and non-organ specific autoantibodies and IgG levels may be used to monitor disease activity in AIH.

A radioimmunoassay for detection of circulating antibodies reacting with the hepatic asialoglycoprotein receptor protein

To determine whether circulating antibodies against the liver-specific membrane lipoprotein complex (LSP), which occur in a number of liver disorders, are directed at the hepatic asialoglycoprotein receptor protein (hepatic lectin)--recently shown to be a constituent of LSP--a radioimmunoassay for anti-hepatic lectin antibodies was developed based on the use of protein A in situ on staphylococcal cells to precipitate 125I-labelled rabbit hepatic lectin/anti-lectin immune complexes. The assay was used to test sera from 30 patients (15 anti-LSP-positive and 15 anti-LSP-negative) with autoimmune chronic active hepatitis (CAH), hepatitis B virus-positive CAH or primary biliary cirrhosis. Anti-lectin antibodies (at titres of 1:200 to 1:1600) were found in all 15 anti-LSP-positive sera and were not detected in the 15 anti-LSP-negative patients. Hepatic lectin is a highly purifiable, liver-specific, hepatocellular surface component and the anti-lectin assay (which proved sensitive, reliable and easy to perform) will permit further exploration of the role of autoimmune mechanisms in the pathogenesis of various liver diseases.

Antibodies to atypical mycobacteria in primary biliary cirrhosis

Recent work has shown that there is antigenic similarity between common bacterial proteins and epitopes on biliary epithelial cells. Following evidence that the sera of all of a series of patients with primary biliary cirrhosis contained antibodies to an extract of Mycobacterium gordonae (a ubiquitous environmental organism) and that these antibodies cross-reacted with the major mitochondrial M2 epitopes, the study below has been performed to investigate this phenomenon further. Using Western blotting of membrane extracts of Mycobacterium gordonae and seven other atypical mycobacterial species, no such antibodies were detected in the serum of patients with primary biliary cirrhosis, but antibodies to a 65 kDa mycobacterial protein were found in most patients with primary biliary cirrhosis as well as in normal controls and patients with other chronic liver diseases.

Auto-immune features in patients with idiopathic chronic active hepatitis who are seronegative for conventional auto-antibodies.

In patients with chronic active hepatitis (CAH), the absence of the conventional serum auto‐antibodies (antinuclear, smooth muscle and liver‐kidney microsomal) is often taken as evidence against an auto‐immune aetiology and as indicative that the disease is unlikely to respond to immunosuppressive therapy. We report 12 British patients (11 female) who presented with histologically florid CAH (11 with cirrhosis or fibrosis and seven with ascites) but without significant titres of these antibodies or any other demonstrable aetiological feature (cryptogenic CAH), who have been followed up for a median of 5.25 years (range: 0.75–16 years). Ten had hypergammaglobulinaemia and/or specific elevations of serum IgG concentrations at presentation and five of 10 patients tested were found to have the HLA allotypes B8 and DR3. Remission was initially induced with prednisolone with or without azathioprine in all patients. Six patients subsequently relapsed on one or more occasions, either spontaneously while on maintenance therapy or during attempts to withdraw corticosteroids, and required increases or reintroduction of immunosuppressive therapy to regain disease control. Retrospective analysis of pretreatment samples from 11 of the patients revealed that all had been seropositive at presentation for auto‐antibodies against the liver membrane lipoprotein preparation known as liver‐specific membrane lipo‐protein (LSP) and/or against the hepatic asialoglycoprotein receptor (ASGP‐R), titres of which subsequently fluctuated in direct relation to response to treatment. The findings suggest that in this group of patients (i) the absence of conventional auto‐antibodies does not exclude the likelihood of a response to immunosuppressive therapy, (ii) auto‐immune mechanisms underlie the disease, and (iii) screening for anti‐LSP and anti‐ASGP‐R may be useful for discriminating them from patients with chronic non‐A, non‐B viral hepatitis.

Cellular and humoral immune reactions against autoantigens and hepatitis C viral antigens in chronic hepatitis C

BACKGROUND/AIMS Previous reports have suggested that the hepatitis C virus (HCV) may induce autoimmune hepatitis. The aim of this study was to examine this hypothesis by investigating humoral and cellular immune responses to HCV-related antigens and various autoantigens in patients with chronic HCV infections. METHODS Lymphoproliferative responses in vitro and/or circulating antibodies to an HCV core peptide, the putative autoantigen GOR, the liver-specific hepatic asialoglycoprotein receptor (ASGP-R), and other autoantigens were investigated in 27 adults with chronic hepatitis C. RESULTS Five patients with HCV (18.5%) showed cellular immune responses to ASGP-R and two others had antibodies to ASGP-R, whereas 6 of 14 patients (42.8%) showed cellular responses to GOR and 7 of 14 patients (50%) showed responses to HCV core. Other autoantibodies were detected in three patients (11%). Nine patients with autoimmune hepatitis studied concurrently for comparison showed cellular and/or humoral responses to ASGP-R but not to GOR. Only 2 of 11 patients with other chronic liver disorders showed immune responses to any antigen tested. CONCLUSIONS Specific immunocompetence against HCV-related antigens can often be shown in patients with chronic hepatitis C but is infrequently accompanied by autoreactions against liver-specific or nonspecific antigens. A reported association between T-cell responses to HCV core and lack of liver damage could not be confirmed.

Development of a micro enzyme-linked immunosorbent assay for antibodies against liver-specific membrane lipoprotein

Guinea pig antisera raised against human and rabbit liver-specific membrane lipoprotein (LSP) have been used to develop a rapid, reproducible and sensitive solid-phase, enzyme-linked immunosorbent assay (ELISA). The ELISA was used in a series of cross-inhibition experiments to define and quantitate the antigenic specificities of these antisera. The results confirm previous findings that LSP contains both liver-specific and liver non-specific antigens. In addition, it is shown that both human and rabbit LSP contain liver-specific antigens that are species-specific, as well as others that are species cross-reactive. Binding to human LSP was detected by the ELISA with 11 of 13 anti-LSP-positive (by radioimmunoassay) sera from patients with HBsAg-negative chronic active hepatitis. Similar binding was also found with 10 of 11 sera from patients with systemic lupus erythematosus and with 7 of 14 with rheumatoid arthritis --all of which were negative for anti-LSP by radioimmunoassay. It is suggested that immune complexes in these sera might bind to IgG-Fc receptors in LSP and that caution should be exercised in the interpretation of data from ELISA-based studies of anti-LSP in human sera.