Development of anti-tissue antibodies in the rat liver transplant model.

BACKGROUND: The aim of this study was to analyze the humoral immune response associated with orthotopic liver transplantation in the rat liver transplant model, and in particular to test the presence of anti-tissue antibodies. METHODS: Rearterialized liver transplantations were performed in the Dark Agouti (DA)-to-Lewis (LEW) and the LEW-to-DA rat strain combinations. Sera of recipients were analyzed by immunofluorescence (on DA and LEW organ sections) and by western blotting (with DA and LEW liver proteins). RESULTS: We have shown that liver (but not heart or skin) recipients develop a humoral response against non-MHC tissue antigens as evidenced (1) by a pattern of staining comparable to that described in human patients harboring anti-smooth muscle antibodies and (2) by the presence of donor liver peptides recognized in the sera of the recipient by Western blotting. CONCLUSIONS: These experiments indicate that orthotopic transplantation of a nonacutely rejected liver allograft is associated with the development of a previously undescribed anti-tissue antibody response that seems to be neither organ nor MHC restricted.

High incidence of antitissue antibodies in patients experiencing chronic liver allograft rejection.

BACKGROUND: The precise immunologic mechanisms responsible for chronic rejection of liver allografts are unknown. We have recently shown in a rodent model that recipients of liver allografts developed non-major histocompatibility complex antitissue antibodies. The aim of the present study was to test this hypothesis in the clinical setting. METHODS: Posttransplant sera of 14 patients undergoing chronic rejection and of 48 control patients (12 liver transplant patients with chronic active hepatitis or liver cirrhosis related to hepatitis C virus [HCV] infection and without chronic rejection, 10 with sclerosing cholangitis, and 26 with normal liver function tests and liver biopsy) were tested for the presence of antitissue antibodies by indirect immunofluorescence. Pretransplant sera of all these patients lacked antitissue antibodies. RESULTS: Antitissue antibodies were detected in 71% of patients who developed chronic rejection (before or at the time of chronic rejection). This incidence was significantly greater than that observed in patients not undergoing rejection (HCV-related chronic active hepatitis, 16%; sclerosing cholangitis, 0%; normal liver biopsy, 7%). All these autoantibodies were directed against the smooth muscle and/or the nucleus. In two patients, anti-smooth muscle antibodies had an antiactin or antivimentin specificity. CONCLUSIONS: These results show a strong association between chronic allograft rejection and the development of antitissue antibodies and suggest that these antibodies could be used to identify patients at high risk of developing chronic rejection after liver transplantation.

Histological features predictive of recurrence of primary biliary cirrhosis after liver transplantation.

BACKGROUND: Recurrence of primary biliary cirrhosis (PBC) within liver allografts remains a controversial issue. The aims of this study were to evaluate this risk and to determine the presence, if any, of a predictive histological feature. METHODS: We reviewed the most recent and the 1-year protocol liver biopsies of 69 patients who received transplants for PBC and of 53 control patients. Histological features consistent with PBC recurrence included nonsuppurative destructive cholangitis, mixed portal infiltrate, fibrosis, and ductopenia. A complete evaluation was undertaken in each patient with these histological features. RESULTS: These histological features were present in six patients who received transplants for PBC (8.7% vs. 0% in the control group) and occurred between 1 and 8 years after transplant. In five of the six patients, anti-mitochondrial antibody-2 (anti-M2) antibodies remained at high titers. Cholestasis was present in four patients, and clinical symptoms in two patients. All six patients were negative for hepatitis C antibodies and hepatitis C RNA in their serum. None had bile duct obstruction. The presence of plasma cells in the portal infiltrate at 1 year after transplant was predictive of this risk of recurrence. CONCLUSION: The risk of PBC recurrence is real (8.7%). The presence of plasma cells in the portal infiltrate seems to be an early marker of recurrence of PBC in patients transplanted for this indication.

[Comparative study of serological markers for intolerance to gluten]. / Etude comparative des marqueurs sérologiques de l'intolérance au gluten.

In France as well as in most of the other European countries, the prevalence of coeliac disease is underestimated. In order to point out a good screening test, we have determined the most sensitive combination (technique-marker) for the diagnosis of the disease among 81 individuals (50 with coeliac disease and 31 controls). Serum anti-gliadin antibodies were measured using three methods: the qualitative dot-blot (Gliastick-Eurospital) and two quantitative methods Elisa (homemade-Saint-Antoine Hospital and alpha-Gliatest-Eurospital); serum anti-endomysium antibodies (EmA) and anti-reticulin antibodies (ARA) were detected using an indirect immunofluorescence assay. We have shown that the simple and fast Gliastick test can fulfil the selected criterion with a sensitivity of 0.90. Nevertheless, uncertain and positives results have to be confirmed by one of the two more specific quantitative tests. The two other markers (ARA and EmA) have shown a better specificity (1) but they were less sensitive (0.54 and 0.56 for ARA and EmA respectively). Thus, they have both to be used as confirmation tests and for follow-up with supervision of the compliance to recommended diet. In conclusion, the Gliastick can be considered as a good screening test for the detection of anti-gliadin antibodies and it would represent the expected help to determine the prevalence of coeliac disease on a large-scale map.

[Analysis of dot-blot technique for the detection of three autoantibodies (anti-Jo-1, anti-M2, anti-ribosome). Comparison with reference techniques]. / Analyse d'une technique de dot-blot pour la détection de trois autoanticorps (anti-Jo-1, anti-M2, antiribosomes). Comparison avec les techniques de référence.

A recently commercialized dot-blot (Cyto-Dot, BMD) offered a new method for the detection of three autoantibodies (Ab) anti-Jo-1, anti-M2, and anti-ribosomal protein although their only common point is the cytoplasmic localisation of their respective antigen. These Ab are detected by indirect immunofluorescence (IF) (anti-M2, anti-ribosomal protein), double immunodiffusion (ID) (anti-Jo-1) and western blotting (WB) (anti-M2). The aim of the study was to compare results obtained by the Cyto-Dot with those obtained by our reference technique. One hundred and seventy-seven sera were analysed, divided into four groups: group I (n = 15) with anti-Jo-1 Ab detected by ID, group II (n = 70) with anti-M2 Ab by WB, group III (n = 33) with anti-ribosomal protein Ab by IF (of which, 19 are precipitating by ID), group IV (control group, n = 59) with 31 sera of healthy individuals, six sera of patients with liver diseases resembling primary biliary cirrhosis and 22 with a particular serological profile. Cyto-Dot is very sensitive and specific for the detection of anti-Jo-1 Ab. Also, it represents a reliable method (sensitivity 0.99) for the screening of anti-M2 Ab and for the confirmation of an atypic immunofluorescence pattern. Equivalent to ID for the detection of anti-ribosomal protein Ab, the Cyto-Dot represents a good alternative technique. However, although this new diagnostic method represents a sensitive technique for the detection of the three auto-Ab, unfortunately, it can not be applied for large series.

[Characterization of anti-mitochondrial antibodies type 2 (anti-M2): comparison of two techniques, western blotting and indirect immunofluorescence]. / Caractérisation des anticorps antimitochondries de type 2 (anti-M2): comparaison de deux techniques, immunotransfert et immunofluorescence indirecte.

About 94% of patients with typical features of primary biliary cirrhosis (PBC) have been shown to be anti-M2 positive. Today the relevance of anti-M2 antibodies as a diagnostic marker of PBC is well established. The usual method of detection is by indirect immunofluorescence with cryostat sections of rat organs. In our laboratory we have developed a second identification technique for these antibodies: Western-blotting. To compare immunofluorescence and immunoblotting results, we selected sera from 252 patients: 142 sera from patients with documented PBC, 50 from patients with another hepatic disease, 10 from patients with haematological lupus and 50 from healthy blood donors. We characterized antimitochondrial antibody M2 by the presence of one or more of five antigenic determinants: 65-70 kDa (a), 52-54 kDa (b), 44 kDa (c), 23-26 kDa (d) and 16-20 kDa (e). This technique is especially useful as a backup method intention for identifying a very slight or atypical fluorescence pattern.

[Autoantibodies in hepatology]. / Auto-anticorps en hépatologie.

The diagnosis of autoimmune liver disease (primary biliary cirrhosis, autoimmune hepatitis, and primary sclerosing cholangitis) can be made by assaying specific autoantibodies in the serum. A review is presented of these antibodies, the methods used to detect them, and the antigens identified to date. Hepatitis C is the most common cause of autoantibody production in liver diseases and is also discussed.

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.

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.

Polyspecificity of antimicrosomal thyroid antibodies in hepatitis C virus-related infection.

OBJECTIVES: The outcome of dysthyroidism and the presence of antithyroid antibodies in patients with chronic hepatitis C virus (HCV) infection receiving interferon-alpha therapy is clearly established. However, the prevalence and the specificity of antithyroid antibodies in HCV patients before interferon-alpha therapy remain controversial. The aim of the present study is to clarify within a large population of HCV patients the prevalence of antithyroid antibodies before interferon-alpha therapy and to determine whether their immunodominant antigen is the same as described in autoimmune thyroiditis. METHODS: Sera from 99 patients with chronic hepatitis C before (n = 99) and after (n = 37) interferon-alpha treatment were investigated for the presence of antimicrosomal and antithyroperoxidase antibodies assessed by indirect immunofluorescence and ELISA, respectively. Dot blotting on human thyroid lysate was designed to further characterize these autoantibodies. Data were compared to those obtained with sera of patients with autoimmune thyroiditis (n = 75) and healthy subjects (n = 96). RESULTS: In HCV patients, antimicrosomal antibodies were found with a higher proportion before interferon-alpha therapy (12.1%) than after therapy (8%). Thyroperoxidase constitutes the main antigen in only 4% before treatment, a prevalence similar to that observed in healthy controls. CONCLUSIONS: The prevalence of antithyroid antibodies is low in patients with chronic hepatitis C before interferon-alpha therapy. Thyroperoxidase may not be their main target. Further studies are required to determine whether HCV infection leads to a breakdown of tolerance to a thyroid self-protein other than thyroperoxidase.

Autoepitope mapping and reactivity of autoantibodies to the dihydrolipoamide dehydrogenase-binding protein (E3BP) and the glycine cleavage proteins in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial antibodies (AMA) directed primarily against the E2 subunits of the pyruvate dehydrogenase complex, the branched chain 2-oxo-acid dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, as well as the dihydrolipoamide dehydrogenase-binding protein (E3BP) of pyruvate dehydrogenase complex. The autoantibody response to each E2 subunit is directed to the lipoic acid binding domain. However, hitherto, the epitope recognized by autoantibodies to E3BP has not been mapped. In this study, we have taken advantage of the recently available full-length human E3BP complementary DNA (cDNA) to map this epitope. In addition, another lipoic binding protein, the H-protein of the glycine cleavage complex, was also studied as a potential autoantigen recognized by AMA. Firstly, the sequence corresponding to the lipoic domain of E3BP (E3BP-LD) was amplified by polymerase chain reaction and recombinant protein and then purified. Immunoreactivity of 45 PBC sera (and 52 control sera) against the purified recombinant E3BP-LD was analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Secondly, reactivity of PBC sera was similarly analyzed by immunoblotting against H-protein. It is interesting that preabsorption of patient sera with the lipoic acid binding domain of E3BP completely removed all reactivity with the entire protein by immunoblotting analysis, suggesting that autoantibodies to E3BP are directed solely to its lipoic acid binding domain. Fifty-three percent of PBC sera reacted with E3BP-LD, with the majority of the response being of the immunoglobulin G (IgG) isotype (95%). Surprisingly, there was little IgM response to the E3BP-LD suggesting that the immune response was secondary because of determinant spreading. In contrast, H-protein does not appear to possess (or expose) autoepitopes recognized by PBC sera. This observation is consistent with structural data on this moiety.

Description of a new DRB1*11 allele (DRB1*1132).

We describe a new DRB1*11 allele which is similar to DRB1*11011 except at codon 74, where a GCG is changed for a GTG leading to an alanine/valine substitution. This new allele was carried by a Caucasian patient suffering from rheumatoid arthritis and by her healthy daughter. The motif at codon 74 of the new DRB1*11 is not found in any other known DRB alleles, nor among the published DQA1, DQB1, DPA1 or DPB1 alleles, and therefore suggests a mechanism of point mutation.