HBsAg-negative Chronic Active Hepatitis Research Articles

Frequency of antibody to hepatitis C virus in asymptomatic HBsAg-negative chronic active hepatitis.

To determine the frequency of antibodies to hepatitis C virus in asymptomatic patients with HBsAg-negative chronic active hepatitis, sera from 30 consecutive patients with few or no symptoms of liver disease were tested by an enzyme immunoassay. The reactivity of antibodies detected by enzyme immunoassay against hepatitis C virus encoded antigens was determined by recombinant immunoblot assay. Antibodies were detected in 11 of the 30 patients (37%) and eight of the seropositive sera (73%) were reactive by recombinant immunoblot assay. Nonreactive patients were weakly positive by enzyme immunoassay (sample/cutoff ratio, less than or equal to 1.9) in contrast to reactive patients (sample/cutoff ratio, greater than or equal to 6.3). The prevalence of immunoserologic markers was similar in patients with and without antibodies (78 vs. 87%) but high titers (greater than or equal to 1:160) were more common in seronegative patients (53 vs. 11%). Additionally, seronegative patients had smooth muscle antibodies (83 vs. 25%, p less than 0.05) and concurrent extrahepatic immunologic diseases (37 vs. 9%) more commonly than seropositive counterparts. We conclude that asymptomatic patients with HBsAg-negative chronic active hepatitis frequently have antibodies to hepatitis C virus. These antibodies commonly react to specific viral antigens, especially if the enzyme immunoassay is strongly positive. Seropositive patients infrequently have concurrent immunologic disorders or smooth muscle antibodies. Immunoserologic markers lack diagnostic specificity except in higher titer.

D-Penicillamine therapy in patients with HBsAg-negative chronic active hepatitis and major prednisolone-induced adverse effects

Six patients with histologically proven HBsAg-negative chronic active hepatitis (CAH), who were initially treated successfully with prednisolone with or without azathioprine, developed unacceptable adverse effects due to prednisolone. In all six patients the liver disease relapsed on reduction of the prednisolone dose and they subsequently entered a trial of low dose D-penicillamine. Two of the patients required early withdrawal of D-penicillamine and a third patient, who had a good clinical and biochemical response initially, developed heavy proteinuria at 14 months. In the remainder, D-penicillamine was well tolerated and the liver disease satisfactorily controlled permitting reduction of the prednisolone dose to 2.5 mg daily. We conclude that in this subgroup of patients with HBsAg-negative CAH and major prednisolone-induced adverse effects, D-penicillamine is an effective alternative therapy although side effects are common.

Low–Dose Corticosteroid Therapy After Multiple Relapses of Severe Hbsag–Negative Chronic Active Hepatitis

To evaluate the efficacy of low-dose corticosteroid therapy after multiple relapses of severe HBsAg-negative chronic active hepatitis, 22 patients who had relapsed on 3.4 +/- 0.4 occasions (range = two to seven relapses) were treated with the lowest dose of medication necessary to ameliorate symptoms and maintain serum AST activity below five-fold normal. Results were compared with those in 31 patients who had received conventional retreatments after 3.4 +/- 0.3 relapses (range = two to eight relapses). During 44 +/- 7 mo of low-dose therapy (range = 9 to 149 mo), one patient (5%) entered sustained remission, 16 patients (72%) continued treatment, two patients (9%) received liver transplantations, two patients (9%) died of liver-related complications and one patient (5%) died of a nonliver-related cause. Drug-related side effects improved in 11 of 13 patients who had acquired them during conventional therapy (85%). The median dose of prednisone was 7.5 mg daily (range = 1 to 17.5 mg) with and without azathioprine. Thirteen patients received long-term treatment consisting of 10 mg or less of prednisone only. Patients receiving conventional treatment entered remission more frequently than those on low-dose therapy (97% vs. 36%, p less than 0.001) but they relapsed after drug withdrawal (53% vs. 87%, p greater than 0.01), required continuous therapy (55% vs. 72%, p greater than 0.1) and died of liver-related complications (10% vs. 9%) as commonly as those receiving low-dose therapy. We conclude that low-dose corticosteroid therapy in patients who have experienced multiple relapses has similar efficacy and less morbidity than conventional retreatments.

Extrahepatic malignancy following long-term immunosuppressive therapy of severe hepatitis B surface antigen-negative chronic active hepatitis.

To determine the frequency, predisposing factors and consequences of extrahepatic malignancy following long-term immunosuppressive therapy of severe HBsAg-negative chronic active hepatitis, 149 patients who had received prednisone (20 mg daily) or prednisone (10 mg daily) in combination with azathioprine (50 mg daily) for at least 6 months were evaluated systematically for 109 +/- 5 months (range: 7 to 223 months). Seven neoplasms involving cervix (2), lymphatic tissue (1), breast (1), bladder (1), soft tissue (1) and unknown site (1) developed in seven patients after 116 +/- 23 months (range: 18 to 164 months). The incidence of extrahepatic neoplasm was 1 per 194 patient-years of surveillance, and the probability of tumor occurrence was 3% after 10 years. Tumor frequency was similar in men and women and the risk was 1.4-fold greater than that in an age- and sex-matched normal population (95% confidence interval, 0.6- to 2.9-fold normal). Patients with extrahepatic malignancy were not distinguished by age, sex, treatment regimen, cumulative duration of treatment (42 +/- 9 vs. 60 +/- 4 months, p = 0.7) or individual features of the liver disease. Five of the seven patients survived during 48 +/- 25 months of follow-up, including two patients who have lived for at least 5 years after the diagnosis of malignancy. We conclude that extrahepatic malignancy develops infrequently during long-term immunosuppressive therapy. Its occurrence is not related to the type or duration of treatment, and long-term survival after tumor detection is possible. The low but probably increased risk of extrahepatic neoplasm does not militate against the use of immunosuppressive therapy in these patients.

CHARACTERISATION OF A NEW SUBGROUP OF AUTOIMMUNE CHRONIC ACTIVE HEPATITIS BY AUTOANTIBODIES AGAINST A SOLUBLE LIVER ANTIGEN

Autoantibodies against a soluble liver antigen (SLA) were detected in 23 patients with HBsAg-negative chronic active hepatitis (CAH) but not in 502 patients with various other hepatic and non-hepatic disorders or 165 healthy blood donors. Anti-SLA-positive serum samples were negative for antinuclear and liver-kidney-microsomal antibodies, markers of two subgroups of autoimmune-type CAH. 6 anti-SLA-positive patients were negative for all autoantibodies sought. Most of the anti-SLA-positive patients were young women (2 men, 21 women, mean age 37 years) with hypergammaglobulinaemia (mean 3·2 g/l, range 1·8-5·3 g/l); 18 of the 23 patients had received immunosuppressive treatment and all responded well. Anti-SLA titres declined during therapy, corresponding to disease activity. Anti-SLA cannot be detected by immunofluorescence. SLA is not organ-specific or species-specific, but the highest concentrations were found in liver and kidney. Anti-SLA autoantibodies characterise a third subgroup of autoimmune-type CAH and will allow a better differentiation of HBsAg-negative CAH which has therapeutic consequences.

Methodology and significance of the detection of liver-kidney-microsomal (lkm) autoantibodies in autoimmune-type chronic active hepatitis

Liver-kidney-microsomal (LKM) autoantibodies are diagnostic markers for a subgroup of HBsAg-negative chronic active hepatitis, presumably owing to autoimmunity. They were originally detected by indirect immunofluorescence and can now be evaluated by radioimmunoassay, enzyme-linked immunosorbent assay, and immunoblotting. In immunoblotting LKM-positive sera react strongly with a 50-kilodalton (KD) polypeptide band of microsomes. In immunoelectron microscopy, LKM-positive sera show a binding with membranes of the endoplasmic reticulum. The LKM antigen was further identified on various isoenzymes of cytochrome P-450. Immunofluorescence is still the method of choice for screening sera routinely. However, cytplasmic antigen-antibody systems often can hardly be distinguished by this method. A specific radioimmunoassay and an enzyme-linked immunosorbent assay can differentiate the various autoantibodies against cytoplasmic antigens. Immunoblotting and immunoelectron microscopy are specific tools for the characterization of the target antigens on an ultrastructural or molecular level. So far they have no use in routine testing of sera. However, since LKM antigen was localized on isozymes of cytochrome P-450, this subgroup of CAH might turn out to be a drug-induced autoimmune liver disease. Clinically, these patients are characterized by chronic active hepatitis or cirrhosis in liver histology, a slight predominance of females, low IgA immunoglobulin levels, and an often rapidly progressing disease. Patients can be treated with immunosuppressive drugs. However, controlled therapeutical trials are missing. Furthermore, an immunogenetic background still has to be proven for this autoimmune liver disease.

HLA typing and autoantibodies in hepatitis B surface antigen-negative chronic active hepatitis.

HLA types, serum autoantibodies and serum globulin levels were surveyed in 46 patients with HBsAg-negative chronic active hepatitis. Patients with chronic active hepatitis with viral risk factors were less likely than those without viral risk factors to be HLA type B8 (10% vs 44%) or to have autoantibodies (antinuclear and/or anti-smooth muscle antibodies) (38% vs 84%). Thirty patients (10 with and 20 without viral risk factors) were treated with glucocorticosteroids. Of the 11 patients who were HLA-B8, 100% responded to treatment. Of the 20 patients who were ANA positive, 80% responded. The data suggest that the presence of HLA-B8 may be a useful predictor of response to anti-inflammatory treatment and may define a genetic subset of HBsAg-negative chronic active hepatitis that will benefit from glucocorticosteroid therapy.

Immunological aspects in patients with chronic active hepatitis--cellular immune responses

We studied host immune parameters which might be related to the activity and the pathogenetic mechanism of chronic active hepatitis. The subjects consisted of 45 cases with hepatitis B virus surface antigen (HBsAg)-positive chronic active hepatitis (CAH), 44 HBsAg-negative CAH, 22 with inactive chronic hepatitis, and 45 cases of normal persons, hepatitis B virus (HBV) carriers, or the patients with acute myocardial infarction. The in vitro assay for the in vivo activated lymphocytes was performed by measuring spontaneous thymidine uptake (SLT) of lymphocytes isolated from peripheral blood. SLT was significantly (p < 0.001) elevated in cases with HBsAg-positive (1227 +/- 806 cpm) and-negative CAH (1017 +/- 559 cpm) compared to the patients with inactive chronic hepatitis (347 +/- 79 cpm) and to the control group (320 +/- 106 cpm). SLT values observed in 7 cases with active disease (group I and II), in which remission and relapsing phase could be assessable, were elevated from 648 +/- 121 cpm in remission phase to 1548 +/- 606 cpm one to two weeks before the appearance of biochemical evidence (SGPT) of relapse. This pattern of SLT elevation, however, was not observed in patients with inactive hepatitis. Neither the abnormal distribution of T-cell subsets nor the presence of conventional HBV markers were related to the elevated SLT value. Our findings may therefore indicate that SLT might be useful in assessment of the disease activity in patients with CAH.(ABSTRACT TRUNCATED AT 250 WORDS)

Gallstone dissolution with ursodeoxycholic acid in patients with chronic active hepatitis and two years follow-up. A pilot study.

Chemical dissolution of cholesterol gallstones using ursodeoxycholic acid (UDCA) in six patients with histologically confirmed HBsAg-negative chronic active hepatitis was started after a minimum of one year of therapy with steroids, azathioprine, or chloroquine and a treatment-free period of 8-15 months. The treatment with UDCA lasted 3-20 months with a daily dose of 8-11 mg/kg. Four patients served as controls. A decrease in transaminases (P less than 0.05) occurred in all patients during the UDCA therapy. After completion of the treatment, the figures rose again, but did not return to the initial value. The stones dissolved in five patients. A second liver biopsy was carried out in two patients after UDCA therapy, and this showed no detectable deterioration. Four patients refused biopsy because the laboratory parameters had improved under UDCA. A stone recurred in one patient six months after the end of therapy; the others have remained free of stones for up to 24 months.

Significance of Serum and Hepatic Markers of Hepatitis B Viral Infection in Hbsag–Positive and Hbsag–Negative Chronic Active Hepatitis

The correlation between serum and hepatic markers of hepatitis B virus (HBV) has been studied in 70 subjects with chronic active hepatitis of whom 18 were HBsAg+ and 52 were HBsAg-. In HBsAg+ subjects, sera were tested for HBeAg/anti-HBe status and for HBV DNA sequences using a DNA dot hybridization technique. Anti-HBs and anti-HBc were measured in serum in the HBsAg- group. Immunoperoxidase staining was used to detect HBsAg, HBcAg and delta antigen in liver tissue. Of the 18 HBsAg+ patients, 13 were HBeAg+ and 5 were anti-HBe+. A good correlation was shown between HBeAg and HBV DNA in serum and HBcAg expression in liver tissue. Neither HBV DNA in serum nor HBcAg in liver tissue was detected in any of the anti-HBe+ patients. HBsAg and/or HBcAg were detected in liver tissue in 17 of 18 HBsAg+ subjects (95%). However, neither HBsAg nor HBcAg were detected in liver tissue in 52 HBsAg- patients. This group included 11 patients with antibody markers in serum of past HBV infection. Thus, in contrast to previous studies, a good correlation was demonstrated between the serum and hepatic markers of viral replication, and no evidence was obtained to implicate the HBV as an etiological agent in HBsAg- chronic active hepatitis.

Mononuclear cell complement receptor blockade in primary biliary cirrhosis.

Peripheral blood monocyte and lymphocyte receptors for Fc and C3b fragments were examined in vitro in patients with primary biliary cirrhosis and other chronic liver diseases using sheep red blood cells coated with anti-SRBC IgG1 (to detect Fc receptors) and with anti-SRBC IgM and complement (to detect C3b receptors). The number of C3b receptors detected on 100 monocytes was significantly lower in patients with primary biliary cirrhosis (23.0 +/- 12.0, mean +/- 1 SD) compared with normal controls (57.4 +/- 16.9) and other chronic liver disease (HBsAg negative chronic active hepatitis 62.0 +/- 17.0, alcoholic cirrhosis 50.9 +/- 4.0), while the number of Fc receptors detected on 100 monocytes was not significantly different in all the groups (primary biliary cirrhosis 72.8 +/- 28.6, chronic active hepatitis 74.7 +/- 14.0, alcoholic cirrhosis 58.0 +/- 13.5 and normal controls 69.6 +/- 19.9). When mononuclear cells isolated from normal individuals were pre-incubated with serum from patients with primary biliary cirrhosis before testing their receptor function there was a significant reduction in the number of C3b receptors detected per 100 monocytes (27.6 +/- 10.8) compared with pre-incubation with normal serum (72.0 +/- 18.0). This reduction in C3b-receptor function was again observed when the serum used for pre-incubation was depleted of circulating immune complexes; but when complement was further depleted from these sera, the number of C3b-receptors detected after pre-incubation was similar to normal values (64.0 +/- 11.8). Lymphocyte receptors showed a similar pattern of results. This implies a specific C3b receptor blockade on monocytes and lymphocytes from patients with primary biliary cirrhosis which appears to be because of blocking by serum factor(s) including complement fragments.

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 of14 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.

Effect of polyunsaturated phosphatidylcholine on immune mediated hepatocyte damage.

Studies were carried out to investigate the mechanisms underlying the reduction of hepatocellular necrosis observed when polyunsaturated phosphatidylcholine was administered to patients with HBsAg negative chronic active hepatitis. After oral administration of the agent, the susceptibility of rabbit hepatocytes to both antibody dependent cell mediated cytotoxicity and mitogen induced lymphocyte cytotoxicity was substantially reduced. Short term in vitro incubation of either the hepatocytes or lymphocytes with polyunsaturated phosphatidylcholine had no effect on antibody dependent cell mediated cytotoxicity. As it has been shown that orally administered polyunsaturated phosphatidylcholine can be incorporated into the liver cell membrane, it is possible that polyunsaturated phosphatidylcholine exerts its effect by blocking the interaction between immune effector cells and hepatocytes.

In vitro synthesis of IgG and IgM in patients with HBsAg-positive and HBsAg-negative chronic active hepatitis.

Spontaneous and PWM-driven IgG and IgM synthesis was investigated in the PBMC of 15 patients with HBsAg-negative CAH and six HBsAg-positive patients with CAH. PBMC from patients with HBsAg-positive CAH show an impaired IgG synthesis upon stimulation with PWM but an IgM increase similar to that of control subjects. In contrast, PBMC from HBsAg-negative patients with CAH show a trend to spontaneous increased synthesis of IgG and a decrease or lack of IgG and IgM synthesis upon PWM stimulation. Steroid treatment seems to ameliorate these alterations. No differences were found among the three groups of HBsAg-negative chronic active hepatitis (autoimmune, HBsAg-related and cryptogenic). These results indicate that differences of B-cell functions may exist in the two groups of CAH patients, not only in spontaneous B-cell activation or PWM-induced Ig synthesis but also in the different classes of Ig.

Cytoskeleton Antibodies in Chronic Active Hepatitis, Primary Biliary Cirrhosis, and Alcoholic Liver Disease

Antibodies to cytoplasmic microfilaments, intermediate filaments (vimentin filaments), and microtubules which comprise the cytoskeleton of the cell were assayed in sera from 23 patients with HBsAg-negative chronic active hepatitis and 1 with HBsAg-positive chronic active hepatitis, 15 patients with primary biliary cirrhosis, 20 patients with alcoholic liver disease, and 32 healthy controls. The cytoskeleton antibodies were assayed by indirect immunofluorescence technique using vinblastine-treated cultured human embryonic fibroblasts as substrates. There was a significantly increased incidence of cytoskeleton antibodies in patients with liver disease as compared to the control group. Antibodies to microfilaments were found frequently in sera from patients with chronic active hepatitis (67%) and primary biliary cirrhosis (53%) but were rare in sera from patients with alcoholic liver disease (25%) and in control sera (3%). On the other hand, antibodies to microtubules were found in 50% of sera from patients with alcoholic liver disease but in only 7 to 13% of sera from other groups. Intermediate filament antibodies of IgG or IgA class were found only in patient sera whereas intermediate filament antibodies of IgM class were found in the majority of sera in all groups including control sera. The highest titers of intermediate filament antibodies were seen in primary biliary cirrhosis and chronic active hepatitis. The production of cytoskeleton antibodies may be due to the reorganization or destruction of cytoskeletal structures in the liver.

Lymphocyte cytotoxicity to autologous hepatocytes in HBsAg positive chronic liver disease.

Lymphocytes from 39 patients with HBsAg positive chronic liver disease were incubated with their own hepatocytes to investigate mechanisms of lymphocyte-mediated liver damage. Cytotoxicity was significantly increased in 46% overall, and in 73% of those with chronic active hepatitis. Unlike HBsAg negative chronic active hepatitis where only non-T cells were cytotoxic, HBsAg positive patients had both cytotoxic T and non-T cells. A purified liver membrane complex (LSP) and aggregated IgG both blocked non-T cytotoxicity without affecting T cell cytotoxicity; this suggests that the former is probably an antibody-dependent cell-mediated reaction against normal membrane components. This was confirmed in preliminary studies which demonstrated that preincubation of hepatocytes with the F(ab)2' fragment of an anti-human IgG reduced non-T lymphocyte cytotoxicity. T-cell cytotoxicity was restricted to HBeAg-positive patients, suggesting a relationship between T-cell cytotoxicity and viral replication. Purified HBsAg, however, blocked cytotoxicity in only three of 11 cases. Non-T lymphocytes reacting with normal membrane components may contribute to liver damage in both 'autoimmune' and virus-associated chronic liver disease, whereas cytotoxic T-cells, probably reacting with viral determinants, are exclusive to those with viral replication.

Oral Pulse Prednisone Therapy in the Treatment of HBsAg Negative Chronic Active Hepatitis

A young woman with hepatitis B surface antigen negative chronic active hepatitis and hypergammaglobulinemia was treated successfully with oral pulse steroid therapy consisting of prednisone, 90 mg/day, given in repeated 3--5-day courses at 3--4-wk intervals. This approach, which is hypothetically founded on the ability of steroids to cause prolonged inhibition of immunoglobulin G synthesis and proposed mechanisms of hepatocellular damage in chronic active hepatitis, permitted complete clinical, chemical, and histologic remission without morbidity. Oral pulse prednisone therapy deserves further study as a possible adjunct or alternative to existing methods of managing hepatitis B surface antigen negative chronic active hepatitis associated with hypergammaglobulinemia.

Lack of cross-reactivity between antibody against smooth muscle and antibodies to measles virus in sera from patients with chronic active hepatitis.

A majority of patients with HBsAg-negative chronic active hepatitis (CAH) show considerably raised titres of serum antibodies against measles virus antigens. Since these patients often have high titers of antiactin specific antibodies against smooth muscle, and since actin is also incorporated into the measles virion during replication, we have examined whether there was any cross-reactivity between the two antigen-antibody systems. Cross-absorptions of sera from patients with CAH were performed using measles virus antigens and smooth muscle from human uterus. The corresponding specific antibodies were removed from serum without significantly affecting the titres of the other antibodies. Absorption had no influence on the total amount of IgG in serum. The raised titres of antibodies against measles virus seen in CAH can therefore not be explained by cross-reaction with antibodies to smooth muscle.

Characterization of measles virus-specific antibodies in sera from patients with chronic active hepatitis.

Measles virus-specific antibodies in sera from patients with HBsAg-negative chronic active hepatitis and raised antibody titres against measles virus, have been examined by crossed immunoelectrophoresis. The immunoprecipitates were further analysed by SDS-polyacrylamide gel electrophoresis. Five measles virus-specific precipitation lines were demonstrated using measles virus-infected cells solubilized with Triton X-100. The three major precipitation lines were analysed by SDS-PAGE and contained the virus polypeptides: nucleoprotein, NP (MW approximately 60 000); haemoagglutinin, H (MW approximately 80 000) and fusion protein F1 (MW approximately 40 000). Considerably higher amounts of antibodies against these three virus polypeptides were demonstrated in the patient sera than in sera from healthy controls. By SDS-PAGE analysis of radiolabelled immune complexes adsorbed to Sepharose-protein A, antibodies against five measles virus polypeptides: NP, H, F1, P protein (MW approximately 70 000) and matrix protein, M (MW approximately 37 000) were demonstrated in the patient sera.

Regulatory T cell-subset imbalance in chronic active hepatitis.

Three monoclonal anti-T-cell antibodies, specifically directed against total T cells (OKT3), inducer-helper T cells (OKT4) and suppressor/cytotoxic T cells (OKT8), were used in this study to analyze peripheral T-cell subsets in hepatitis B surface antigen (HBsAg)-positive and -negative chronic active hepatitis (CAH) patients. Results showed that a clear-cut difference exists in the distribution of peripheral T cells of these two groups of subjects. HBsAg-positive CAH patients had a numerical predominance of peripheral T lymphocytes expressing the characteristics of cytotoxic/suppressor T cells. In contrast, patients with "autoimmune" HBsAg-negative CAH exhibit a predominance of OKT4 cells, namely, the helper-inducer T-cell subset. In addition, high numbers of circulating double labeled cells (expressing both the OKT4 and the OKT8 xenoantigens) were detected in some of the HBsAg-positive and HBsAg-negative CAH patients studied.