Outcome of orthotopic liver transplantation in autoimmune hepatitis according to subtypes.

The relevance of autoimmune hepatitis (AIH) classification for clinical purposes is controversial. We analyzed the outcome after orthotopic liver transplantation (OLT) of nine type I and seven type II AIH patients. Type II patients had a significantly higher incidence of cirrhosis at the time of diagnosis, more resistance to steroid therapy, and a higher Child-Pugh score at the time of OLT. OLT was performed in emergency in three type II patients and electively in all type I patients. Four type II and one type I patients died in the postoperative period. There was no difference regarding the incidence of post-OLT infection and rejection between the two types. No recurrence of AIH was observed. The 6-year actuarial survival rates for type I and type II patients were 76% and 43%, respectively. Type II AIH patients who have a poor response to medical therapy should be considered for OLT with a shortened delay.

Antibodies to soluble liver antigen: an additional marker in type 1 auto-immune hepatitis.

BACKGROUND/AIMS: Auto-immune hepatitis patients are divided into two well-defined subgroups on the basis of immunoserological markers, i.e. anti-actin cable and/or anti-nuclear antibodies for the auto-immune hepatitis type 1, anti-liver/kidney microsome type 1 and/or anti-liver cytosol type 1 for the autoimmune hepatitis type 2. Controversial antibodies to a soluble liver antigen have been proposed as a diagnostic marker for the putative auto-immune hepatitis type 3. The aim was to investigate the implication of anti-soluble liver antigen antibodies in the diagnosis of auto-immune hepatitis and their ability to define auto-immune hepatitis type 3. METHODS: Sera from 483 patients with hepatic and non-hepatic diseases, and 102 sera from blood donors were analyzed by an inhibition capture enzyme-linked immunosorbent assay. RESULTS: Anti-soluble liver antigen antibodies were found in 13 of the 106 (12%) auto-immune hepatitis type 1 patients and 10 of the 49 (20%) cryptogenic hepatitis patients tested. In contrast, they were not detected in auto-immune hepatitis type 2 (n=54), primary sclerosing cholangitis (n=37), primary biliary cirrhosis (n=52), hepatitis C virus infection (n=105), alcoholic hepatitis (n=25), various non-hepatic autoimmune disorders (n=55) and in healthy blood donors (n=102). The clinical and biological features of antisoluble liver antigen-seropositive patients were similar to those of auto-immune hepatitis type 1 and did not distinguish a subgroup of auto-immune hepatitis. CONCLUSION: The data support the concept that antisoluble liver antigen-positive cryptogenic hepatitis is similar to auto-immune hepatitis type 1. Anti-soluble liver antigen antibodies can be considered as an additional and specific auto-immune hepatitis type 1 diagnostic marker.

[Drug-induced autoimmune hemolytic anemia]. / Anémies hémolytiques autoimmunes dues à des médicaments.

AUTOANTIBODY PRODUCTION: The production of autoantibodies can only occur if immune tolerance is circumvented. Thus drug-induced autoimmune hemolytic anemia requires that the drug have an effect on both autoantigens and on the immune system. AN EXAMPLE, METHYLDOPA: Methyldopa is a hypotensive agent which induces major production of anti-Rh IgG anti-erythrocyte autoantibodies, anti-nuclear antibodies and anti-actin antibodies. These autoantibodies generally appear 6 months after treatment onset and are observed in 20% of treated patients. Hemolysis is however exceptional and is only clinically or biologically perceptible in 1 to 2% of the patients who become immunized. Induced lupus has been reported as have been several dozen cases of drug-induced hepatitis with anti-actin autoantibodies. DRUGS INDUCING HEMOLYTIC ANEMIA: Besides methyldopa, other drugs known to induce hemolytic anemia include levodopa used for Parkinson's disease, mefenamic acid, a nonsteroidal antiinflammatory drug, interferon-alpha, used in chronic viral hepatitis, cyclosporin used for the prevention of graft rejection and the treatment of certain autoimmune diseases, and fludarabin, used in chronic lymphoid leukemia. THERAPEUTIC STRATEGY: If there is no clinical or biological expression, the drug can be continued, excepting fludarabin where regular controls are needed. If hemolytic anemia is patent, the drug must be discontinued, transfusion and corticosteroid therapy should be envisaged.

Formiminotransferase cyclodeaminase is an organ-specific autoantigen recognized by sera of patients with autoimmune hepatitis.

BACKGROUND & AIMS: Anti-liver cytosol type 1 autoantibodies have been reported in association with anti-liver-kidney microsome type 1 autoantibodies in 30% of patients with autoimmune hepatitis type II. In 10% of cases, anti-liver cytosol type 1 antibodies are the only liver-related circulating autoantibodies. The liver cytosol antigen is a liver-specific 62-kilodalton protein present in the cell as an oligomer of approximately 240 kilodaltons. The aim of this study was to identify the antigen recognized by anti-liver cytosol antibody. METHODS: To identify the liver cytosol antigen, an anti-liver cytosol type 1-positive serum was used for the screening of a complementary DNA library from HepG2 cells. Double immunodiffusion method was used to show the identity between the cytosolic and the cloned protein. RESULTS: The sequence of two isolated clones showed 85.2% homology with the formiminotransferase cyclodeaminase (FTCD) enzyme from pig liver. Antibodies purified by affinity with the recombinant protein and sera from mice immunized with FTCD recognized a 62-kilodalton human cytosolic protein when tested by immunoblot. The identity of precipitation lines was found between the cytosolic antigen and FTCD. CONCLUSIONS: This enzyme is a liver-specific antigen recognized by the sera of patients with autoimmune hepatitis.

A new approach to cytochrome CYP2D6 antibody detection in autoimmune hepatitis type-2 (AIH-2) and chronic hepatitis C virus (HCV) infection: a sensitive and quantitative radioligand assay.

Antibodies specific for cytochrome CYP2D6, formally known as liver-kidney-microsome type-1 antibodies (LKM-1), are characteristically found in a subgroup of patients presenting autoimmune hepatitis. They are also found in some patients with chronic HCV infection. These autoantibodies are usually detected by indirect immunofluorescence, immunoblotting and ELISA tests. In an attempt to set up a more sensitive detection assay we developed a quantitative immunoprecipitation radioligand assay using a 35S-methionine-labelled CYP2D6 antigen obtained by in vitro transcription and translation synthesis. All 16 sera from AIH-2 patients strongly bound to this CYP2D6 antigen. Two of the nine sera (22%) from AIH-2 patients that presented only liver cytosol-1 antibodies also bound to CYP2D6. All 24 sera from HCV patients that were positive for LKM-1 antibodies by indirect immunofluorescence were also positive using this CYP2D6 radioligand assay. Lastly, all 15 sera from HCV patients negative for LKM-1 antibodies were negative by this test. The present results support the view that this quantitative radioligand assay is more sensitive than immunoblotting and ELISA CYP2D6 assays, and that it could be used in combination with indirect immunofluorescence assay.

Study of the B cell response to cytochrome P450IID6 in sera from chronic hepatitis C patients.

Some patients with chronic hepatitis C develop liver/kidney microsome type 1 antibodies. Some of these autoantibodies are directed against the cytochrome P450IID6. The aim of this study was to characterize the immunogenic sites on the P450IID6 molecule recognized by autoantibodies from individuals infected with the hepatitis C virus. Serum from 24 patients with markers of hepatitis C infection and liver/kidney microsome type 1 antibodies were tested by immunoblotting with human liver microsomal proteins. Eight of them with anti-P450IID6 antibodies were tested with synthetic peptides representing P450IID6 putative immunogenic sites and for their capacity to inhibit in vitro P450IID6 enzymatic activity. Anti-P450IID6 antibodies in the sera of chronic hepatitis C patients were of IgG subclass 1 and in one patient also of IgM type. These sera recognized different P450IID6 peptide sequences consisting of amino acids (aa) 200-214 and aa 271-339. The synthetic peptide between aa 321 and 339 appears to represent the main antigenic site. Five out of eight anti-P450IID6 positive sera were also able to inhibit P450IID6 enzymatic activity in vitro at a dilution of 1:100. The anti-P450IID6 autoimmune response in chronic hepatitis C patients is polyclonal, probably inducible, and maintained by the liberation of P450IID6 by hepatocyte lysis. The characterization of the P450IID6 immunogenic site recognized by patients with hepatitis C infection may enable a specific test to be designed to identify those patients with autoimmune hepatitis type 2.

Antimitochondrial antibodies in patients with chronic hepatitis C.

Although autoantibodies have been found in the serum of patients with chronic hepatitis C virus (HCV) there has been no convincing evidence of the presence of antimitochondrial antibodies, until now. Sera from 460 untreated patients with chronic hepatitis C were tested for antimitochondrial antibodies, using an indirect immunofluorescence technique; and if they tested positive for the antibodies (titer more than 1:50), they also were treated by Western blot analysis. Seven (1.5%) sera were positive. None of the patients had biological or histological evidence of primary biliary cirrhosis. Antimitochondrial antibodies recognized one of the oxo-dehydrogenase multienzyme complexe's epitopes by Western blot assay in three patients only. All seven patients were then treated by interferon alpha for six months. None showed exacerbation of liver disease during treatment. HCV-RNA disappeared from the serum in one patient who became negative for anti-M2 antibodies. The four patients who did not respond to interferon-alpha therapy, and the two who relapsed after treatment withdrawal, had sustained positive antimitochondrial antibodies. These data suggest that: 1) antimitochondrial antibodies present in patients with chronic hepatitis C do not always recognize the same epitopes as in primary biliary cirrhosis; 2) these antibodies may disappear after eradication of HCV, suggesting that the production of antimitochondrial antibodies is linked to the presence of the virus and 3) the clinical and biological course of chronic hepatitis C, and the response to interferon-alpha therapy, does not seem to be different in patients who are positive for antimitochondrial antibodies.

Specificity and sensitivity of gp210 autoantibodies detected using an enzyme-linked immunosorbent assay and a synthetic polypeptide in the diagnosis of primary biliary cirrhosis.

Between 10% and 42% of patients with primary biliary cirrhosis (PBC) have been reported to have autoantibodies directed against a restricted epitope of gp210, a glycoprotein of the nuclear pore membrane. The prevalence and specificity of these antibodies was studied in a French series of 285 patients with PBC and 497 control individuals affected with other liver or autoimmune diseases. Sera were analyzed by an enzyme-linked immunosorbent assay (ELISA) that used a synthetic polypeptide containing the predominant autoepitope of gp210, in parallel to immunoblotting of gp210 protein and immunofluorescence microscopy. Autoantibodies to the gp210 epitope detected by ELISA were 25.5% sensitive and 99.5% specific for the diagnosis of PBC. These results were in agreement with a 99.4% specificity with immunoblotting analysis and a 96.6% specificity with immunofluorescence. In a subset of PBC patients without detectable antimitochondrial autoantibodies (AMA), gp210 autoantibodies were found in 7 of 15 patients (47%). Therefore, gp210 autoantibodies are highly specific for PBC and may be of particular utility in assessing patients without AMA or with other atypical presentations.

Antineutrophil cytoplasmic autoantibodies in autoimmune liver and inflammatory bowel diseases.

Perinuclear antineutrophil cytoplasmic autoantibodies have been described in inflammatory bowel diseases and in primary sclerosing cholangitis. Because the data concerning their occurrence are conflicting, we have used indirect immunofluorescence on ethanol-fixed neutrophils to test the sera from a large population of 382 patients with various liver and digestive diseases: in particular, from 27 patients with primary sclerosing cholangitis, 105 patients with autoimmune chronic active hepatitis, 30 patients with primary biliary cirrhosis and 124 patients with inflammatory bowel disease. The prevalence of the perinuclear antineutrophil cytoplasmic autoantibodies was 37% in ulcerative colitis and 15% in Crohn's disease. They would not be helpful in the differential diagnosis between these two inflammatory bowel diseases. Within the group of autoimmune liver diseases, perinuclear antineutrophil cytoplasmic autoantibodies were detected in 44% of sera from patients with primary sclerosing cholangitis and in 36% of sera from patients with type I autoimmune active hepatitis, but not in primary biliary cirrhosis. When primary sclerosing cholangitis was associated with an inflammatory bowel disease, the prevalence of these autoantibodies was 60%. They were 88% specific for primary sclerosing cholangitis and 86% specific for type I autoimmune active hepatitis. Despite their moderate sensitivity and specificity in primary sclerosing cholangitis, they remain the only serologic marker of this autoimmune liver disease. Moreover, they turned out to be a more sensitive marker for inflammatory bowel disease with associated primary sclerosing cholangitis.

Human anti-mitochondria autoantibodies appearing in iproniazid-induced immunoallergic hepatitis recognize human liver monoamine oxidase B.

Anti-mitochondria (anti-M6) autoantibodies have been found in the serum of patients with immunoallergic iproniazid (Marsilid)-induced hepatitis, but to date the identity of the protein antigen has not been determined. Here we show, using immunoprecipitation of pargyline-labelled proteins, that among the mitochondrial proteins, liver MAO-B is specifically recognized by the sera containing anti-M6 antibodies. Moreover the enzymatic activity of MAO-B towards phenylethylamine and tyramine is also suppressed after this immunoprecipitation, contrary to the MAO-A activity towards 5-hydroxy-tryptamine. As MAO is irreversibly inhibited by iproniazid, these results suggest that the mechanism of iproniazid-induced appearance of anti-M6 antibodies could be another example of the reactive metabolite/enzyme haptenization mechanism already proposed in the case of tienilic acid for the appearance of anti-organelle antibodies in a drug-induced hepatitis.

Kinetics of anti-M2 antibodies after liver transplantation for primary biliary cirrhosis.

BACKGROUND/AIMS: Orthotopic liver transplantation is currently considered as the treatment of choice for primary biliary cirrhosis in the terminal stage and, as for other autoimmune liver disease, the risk of recurrence of the disease within the graft has been raised. There is, however, some discrepancy about the risk of recurrence based on pathological analysis. In addition, pathological recurrence of primary biliary cirrhosis within the graft is not always associated with a rise in the serological markers of the disease. In order to clarify this situation, we have monitored antimitochondrial antibodies before and after transplantation. METHODS: Antimitochondrial antibodies were detected by indirect immunofluorescence (variation in antibody titers) and the antimitochondrial antibodies-2 by western blotting (variation in the number of peptides recognized in 16 primary biliary cirrhosis patients followed for at least 4 years after transplantation. RESULTS: Antimitochondrial antibody titers had normalized 1 year after transplantation in seven patients, declined in seven others and remained unchanged in two. Over the 4 years of follow up, four patients demonstrated a subsequent increase in antimitochondrial antibody titers. Western blot analysis demonstrated the loss of one or more bands in seven patients during the first operative year after transplantation and in three other patients thereafter; in six patients the western blotting profile remained identical to that obtained before transplantation. The important changes generally occurred during the first year post-transplantation, without significant changes thereafter, except for three patients who demonstrated a secondary reappearance of the initially lost band. Disappearance of all bands was never observed. There was no concordance between the normalization of antimitochondrial antibody titers (indirect immunofluorescence) and the reduction in the number of peptides recognized (western blotting). Serum bilirubin and alkaline phosphatase levels had normalized by 1 year after transplantation, and remained normal thereafter. Routine liver biopsies performed on a yearly basis did not disclose any pattern suggestive of primary biliary cirrhosis recurrence. CONCLUSIONS: Antimitochondrial antibody titers decreased in primary biliary cirrhosis patients after liver transplantation, although antimitochondrial antibodies-2 never disappeared as assessed by western blotting. In the present study these features were not associated with biochemical or histological (correction of histoclogical) evidence of primary biliary cirrhosis recurrence.

Absence of anti-LKM-1 antibody in hepatitis C viral infection in the United States of America.

Several studies from Europe have observed a relationship between hepatitis C virus infection and anti-liver/kidney microsome-1 (anti-LKM-1) positive chronic hepatitis. It has been suggested that hepatitis C may induce an autoimmune phenomenon that leads to the development of a specific type (type II anti-LKM-1 positive) autoimmune chronic hepatitis. We evaluated 204 sera from patients with well-documented hepatitis C infection from two centres in the United States of America and compared them with sera from 428 French patients from three centres. We evaluated the serological prevalence of anti-smooth muscle antibodies, anti-nuclear antibodies, anti-liver cytosol antibodies, and anti-mitochondrial antibodies subtype anti-M2 in patients with chronic hepatitis C. The two groups were matched in their ages, gender, mode of transmission of hepatitis C infection and severity of liver disease. Anti-LKM-1 was not observed in the patients from the USA at a time when it was noted in 3.7% of French patients. There were no differences, however, in the expression of other auto-antibodies, which were often in low titres. Absence of anti-LKM-1 in USA sera in comparison with French sera suggests that there may be differences in induction of anti-LKM-1 related to environmental and/or host genetic factors, and/or genomic variation in the hepatitis C virus.

Association of antimitochondrial antibodies type 5 and anti-beta 2 glycoprotein I antibodies in the antiphospholipid syndrome.

OBJECTIVE: To evaluate if antimitochondrial type 5 antibodies (AMA5) might be included among antiphospholipid syndrome (APS) markers. METHODS: In a retrospective study, blood variables of 48 patients with AMA5 were analyzed in relationship with clinical and biological markers of APS and systemic lupus erythematosus (SLE). RESULTS: We observed a high prevalence of false biological test for syphilis (95%), lupus anticoagulant (LAC) (71%), anticardiolipin antibodies (aCL) of IgG (71%) and IgM (75%) isotype, positive direct Coombs' test (54%), thrombocytopenia (52%), anti-B2 glycoprotein I antibodies (38%). Twenty-nine patients (61%) had at least one clinical manifestation of APS; 42% had recurrent arterial and/or venous deep thrombosis and 21% had recurrent fetal loss. But, for 2 patients, AMA5 were the sole detected immunological marker. Moreover, SLE was observed in 35% of the patients. These were different from 100 control patients with SLE with the respect to skin involvement and dsDNA antibodies which were less frequent (p < 0.01) and aCL, LAC, false biological test for syphilis (p < 0.001), positive direct Coombs' test and thrombocytopenia (p < 0.05) which were more frequent. CONCLUSION: Our data suggests (1) AMA5 is another marker of the APS (2) in patients with SLE, AMA5 seems to be a marker of a subset of SLE. This appears to justify the routine detection of these antibodies.

A peripheral protein associated with the cis-Golgi network redistributes in the intermediate compartment upon brefeldin A treatment.

Human autoantibodies offer unique tools for the study of cellular constituents since they usually recognize highly conserved components, the most difficult to detect due to their low immunogenicity. The serum from a patient with Sjögren's syndrome (RM serum) showing a very high reactivity to the Golgi complex has been shown to immunoprecipitate and to immunodetect by Western blotting experiments a protein mol wt 210,000 (p210) that was shown to be peripheral and cytoplasmically disposed. A close examination of the p210 labeling revealed some differences with Golgi markers: RM serum staining was slightly more extensive than several Golgi markers and showed a discontinuous or granular appearance. Nocodazole induced a specific and early segregation of many p210-associated vesicles or tubules from Golgi apparatus. Upon brefeldin A treatment, p210 did not redistribute in the ER as did other Golgi proteins. In contrast, it exhibited a vesicular pattern reminiscent to that displayed by proteins residing in the intermediate compartment. Double staining immunofluorescence using the RM serum and the marker of the intermediate compartment, p58, revealed segregation of both proteins in control conditions but colocalization in BFA-treated cells. We have further demonstrated by combining different drug treatments that p210-containing elements in brefeldin A-treated cells belong indeed to the intermediate compartment. Experiments on brefeldin A recovery suggested that these p210 elements might play a role in reformation and repositioning of the Golgi apparatus. Ultrastructural localization performed by immunoperoxidase staining allowed us to establish that p210 interacted with the external side of an abundant tubulo-vesicular system on the cis side of the Golgi complex which extended to connecting structures and vesicles between saccules or stacks of cisternae, p210 appears to be a novel protein residing in the cis-Golgi network that may cycle between the Golgi apparatus and the intermediate compartment.

[Anti-mitochondrial antibodies type 5 are markers of antiphospholipid syndrome]. / Les anticorps antimitochondries de type 5 sont des marqueurs du syndrome des antiphospholipides.

In order to investigate the clinical value of anti-mitochondrial antibodies type 5 (anti-M5), we carried out a retrospective study on 48 patients with these antibodies. Seventeen of these 48 patients (35%) satisfied at least 4 criteria of the revised American Rheumatism Association classification of SLE. Twenty-nine (61%) had at least one clinical manifestation of anti-phospholipid syndrome; thirteen had symptoms consistent with primary anti-phospholipid syndrome; five had isolated recurrent thrombosis; five had Evans' syndrome; four had auto-immune haemolytic anaemia; two had immunologic thrombocytopenia. Two of the 48 patients had no clinical manifestations, but only anti-M5 and a false laboratory test for syphilis (FBTS). Our data confirm that patients with anti-M5 have a high prevalence of: 1) thrombosis (42% had three or more deep thromboses) and fetal loss (21%); 2) auto-immune cytopenia with idiopathic thrombocytopenic purpura (29%) and auto-immune haemolytic anaemia (54%); 3) laboratory markers of anti-phospholipid syndrome (lupus anticoagulant (71%), FBTS (95%) and anticardiolipin antibodies (aCL) (71%). For 32 patients with anti-M5, anti-beta 2 glycoprotein I antibodies were also tested; 12 (38%) were positive, all of whom had IgG aCL, ie none had anti-beta 2GPI antibodies without aCL. There was no association between the presence of anti-beta 2GPI antibodies and recurrent thrombosis among patients with anti-M5. All these findings suggest that anti-M5 is another marker of the antiphospholipid syndrome. Even though the prevalence of anti-M5 is low, especially in SLE, it was the only marker of the anti-phospholipid syndrome in two patients; this appears to justify routine screening for these antibodies.

[Prevalence and significance of non-specific anti-organelle antibodies of the liver in chronic viral hepatitis C]. / Prévalence et signification des anticorps anti-organites non spécifiques du foie au cours des hépatites chroniques virales C.

OBJECTIVES: The aim of this study was to evaluate the prevalence and the clinical signification of non organ specific autoantibodies in chronic hepatitis C. METHODS: We studied retrospectively 158 consecutive patients (97 with chronic hepatitis C, 24 with chronic hepatitis B, 67 with alcoholic cirrhosis) and 100 blood-donors. RESULTS: The prevalence of anti-nuclear and anti-smooth muscle antibodies was lower in blood donors than in patients (P < 0.001), but was comparable among the 3 groups of patients. The anti-liver-kidney microsome type 1 antibodies were detected only in patients with chronic hepatitis C (6%). The serum gammaglobulin level was significantly higher in patients with hepatitis C and anti-nuclear antibody titers > or = 1/50. The anti-smooth muscle antibodies detected in patients with hepatitis C had no anti-actin specificity. The response to interferon was not related to the detection of non organ specific autoantibodies before treatment. CONCLUSION: Anti-nuclear or anti-smooth muscle antibodies are not characteristic of hepatitis C virus infection.