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Background: In a recent study we had evidence that sulphite oxidase (SO) may be a relevant autoantigen in primary sclerosing cholangitis (PSC). Aim of the present study was, therefore, to analyse humoral and cellular immune-reactivity towards SO in these patients in more detail., Methods: Sera from 53 patients with PSC (30 untreated and 23 treated with ursodeoxycholic acid [UDCA] at time of analysis), from 422 patients with different hepatic and non-hepatic disorders, and from 50 healthy individuals were tested by ELISA for antibodies against full-length-SO (SO-fl) and its three major domains expressed in E.coli (SO-I, SO-II, SO-III). For epitope-mapping, 29 overlapping peptides were used. Peripheral blood mononuclear cells (PBMC) were obtained from 33 PSC-patients and analysed for SO-induced proliferation, production of cytokines, and expression of the activation marker cluster of differentiation (CD) 69., Results: 43% of the 30 untreated and 26% of the 23 treated PSC-patients had IgG anti-SO-antibodies predominantly reacting with SO-fl, SO-I and SO-II. Antibody-reactivity decreased after UDCA-treatment. Prevalence and reactivity of anti-SO-antibodies were significantly higher in PSC than in patients with other hepatic and non-hepatic disorders. Epitope mapping revealed no distinct immuno-dominant regions within SO. Incubation of PBMC from PSC-patients (but not from controls) with SO-antigens revealed an activation of B-cells and a T-helper cell type-2 reaction pattern (production of interleukin [IL]-13, IL-10)., Conclusions: PSC-patients show humoral and cellular immune response towards SO. Antibodies may be predominantly directed against conformational epitopes. SO enhances in vitro especially T-helper cell type-2 immune-reactions, which may be pro-fibrotic. SO is a detoxifying enzyme present also in bacteria; further studies analysing its role in the aetiology and pathogenesis in PSC may, therefore, be important.

Increase of liver enzymes during therapy with endothelin receptor antagonist (ERA) because of pulmonary arterial hypertension (PAH) has been observed quite frequently the cause of which is unknown. Here we describe a female patient who suffered from autoimmune hepatitis (AIH) type I [positive for antinuclear (ANA) and antiactin antibodies] who developed systemic sclerosis (SSc) with PAH. AIH was treated with corticosteroids and azathioprine, and PAH with the ERA sitaxentan. Reactivation of AIH was observed in the course of therapy with sitaxentan as shown by an increase of liver enzymes, immunoglobulin G globulins, the reappearance of antinuclear and antiactin antibodies and the induction of a further AIH marker antibody reacting with the soluble liver/liver pancreas antigen. Therapy with ERA for pulmonary hypertension may increase the risk for development or exacerbation of AIH.

Background: A high incidence of autoantibodies to lens proteins has been found in sera of patients with uveitis. We showed previously that the anti-lens antibodies reacted predominantly with α-crystallins. The aim of the present study was to identify immunodominant epitopes within the protein chains of human αA- and αB-crystallin., Methods: Epitope specificities of antibodies to αA- and αB-crystallin were examined by ELISA using synthetic overlapping peptides, spanning the entire length of both α-crystallins. The peptides consisted of 25 amino acid residues, with an overlap of at least eight amino acids each. The synthetic peptides were tested against sera of 110 patients with different uveitis forms, classified according to anatomical location of intraocular inflammation., Results: Four immunodominant regions within the protein chains of αA- and αB-crystallin could be identified. These regions were recognized by antibodies in sera of 56% of uveitis patients. Anti-lens antibodies of IgG-type reacted preferentially with regions located at amino acid (aa) residues aa:69-93 and aa:137-161 of αA-crystallin as well as aa:69-110 and aa:137-161 of αB-crystallin. IgM antibodies recognized predominantly region aa:149-173 of αA-crystallin, and aa:69-110 and aa:151-175 of αB-crystallin. IgM antibodies directed to peptide aa:69-93 of αB-crystallin were found in sera of 30% of patients with intermediate uveitis., Conclusions: Four immunodominant B-cell epitopes within the protein chains of αA- and αB-crystallin have been identified; however, no clear correlation with the anatomically defined uveitis subtypes has been found except for intermediate uveitis. Whether there may be a correlation with uveitis forms with similar etiopathogenesis has to be evaluated in further studies.

Background and Aims: Recently, a non-M2-related mitochondrial 60 kDa protein found to be recognized by antimitochondrial antibody (AMA) negative sera from patients with primary biliary cirrhosis (PBC) has been shown to contain parts of the five F(1)-ATPase subunits α, β, γ, δ and ε. In this study, we examined whether this enzyme is, indeed, a target antigen in PBC., Methods: Analysed were 60 AMA-positive/anti-M2-negative and 103 anti-M2-positive PBC patients, 46 patients with autoimmune hepatitis (AIH), 35 patients with primary sclerosing cholangitis (PSC), 110 patients with viral hepatitis, 40 patients with inflammatory bowel diseases (IBD), 33 patients with connective tissue diseases (systemic lupus erythematosus, mixed connective tissue disease, Sjögren disease, systemic sclerosis) and 25 blood donors. The F(1)-ATPase-subunits α-δ were recombinantly expressed in Escherichia coli, purified and applied to ELISA and Western blotting., Results: In all, 40 of the 60 AMA-positive/anti-M2-negative (67%) and 44 (43%) of the 103 anti-M2-positive PBC-sera reacted with at least one of the F(1)-subunits α-δ. The β- and γ-subunits were preferentially recognized. However, also up to 57% of patients with AIH and 34% of patients with PSC had anti-β- or γ-antibodies, while patients with viral hepatitis had these antibodies in up to 13%. Patients with IBD had anti-β and anti-γ-antibodies in up to 20 and 5% respectively. None of the patients with connective tissue diseases had antibodies to the β- and only 6% to the γ-subunit. Sera from healthy blood donors were negative., Conclusions: Antibodies to the β- and γ-subunits of F(1)-ATPase are further AMAs in PBC but occur also in other autoimmune liver disorders; they may be, therefore, indicators for a general autoimmune process of the liver., (© 2011 John Wiley & Sons A/S.)

In a previous study we found in 50% of patients with neuropsychiatric manifestations of systemic lupus erythematosus (NP-SLE) organ specific antibodies to 45-56 kD proteins in a 100,000 g supernatant (SN) from bovine brain mitochondria. Aim of the present study was to identify the corresponding target antigen. A 100,000 g SN from bovine brain mitochondria was applied to SDS-gel electrophoresis. A 50 kD band recognized by sera from patients with NP-SLE in the Western blot (WB) was excised from the gels and applied to mass spectrometry. The identified protein was expressed in Escherichia coli and retested against sera from eleven patients with NP-SLE (severe symptoms n=6, mild symptoms n=5), 26 SLE-patients without NP manifestations and 53 controls (patients with multiple sclerosis, epilepsy, healthy blood donors). Mass spectrometry of the 50 kD band revealed the presence of α-tubulin. Applying the recombinant α-tubulin in the WB, four of the eleven NP-SLE patients (36%), one of the 26 patients with SLE without NP manifestations (4%) and none of the 53 controls reacted with α-tubulin. The antibodies were more frequently found in patients with severe (50%) than with mild NP-SLE (20%). α-tubulin may be a novel marker autoantigen for a neuropsychiatric manifestation at least in a subgroup of patients with SLE. Whether anti-α-tubulin antibodies are of pathogenetic relevance has still to be clarified., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Uveitis is an intraocular inflammatory disease in which autoimmune reactions have been discussed in playing an important role. Many data in this respect derived from the animal model of "experimental autoimmune uveitis", where several organ-specific autoantigens have been described such as the retinal S-antigen and the interphotoreceptor retinoid-binding protein. However, their diagnostic and pathogenic role in humans has been controversially discussed. In recent studies, the possible relevance of betaB1-crystallin, present in lens and ciliary body, has been outlined. We, therefore, wanted to analyse whether sera from patients with uveitis might contain antibodies to lens proteins, Methods: Human lenses from cadaveric eyes were shock frozen, homogenized, and resuspended. The resulting suspension (human lens protein fraction--HLPF) was analyzed for antigenicity by ELISA and Western blotting with patients' sera. A total of 165 patients with uveitis, 54 patients with scleritis and episcleritis, 56 patients with other eye diseases, and 112 healthy blood donors were studied., Results: Twenty-six (49%) of the 53 patients with anterior uveitis, 17 (32%) of the 53 patients with intermediate uveitis and 7 (22%) of 32 patients with posterior uveitis reacted in the ELISA with the HLPF. Antibodies to lens antigens were detected in one-third of patients with panuveitis and retinal vasculitis. In contrast, only 12% of the healthy blood donors were positive in the ELISA. The number of patients with an autoimmune response to alpha-crystallins in Western blot predominated in all investigated groups., Conclusions: These data indicate that antibodies to lens proteins occur in a high incidence in sera from patients with uveitis. Forty-nine percent of the patients with anterior uveitis and only 12% of healthy controls were positive in the ELISA. In our groups of patients and controls the autoantibodies reacted in the Western blot predominantly with alpha-crystallin. Further studies are required to analyze in more detail the clinical and etiopathogenetic relevance of the antilens antibodies in uveitis.

Background: Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen., Patients and Methods: M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive (n=5) or negative (n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC)., Results: Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2(+)/E1(+) and the CFT-negative sera as PDC-E2(+)/E1(-). The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1alpha and E1beta antibodies at a frequency of 74% and 67%., Conclusions: CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target.

Background and Aims Recently, a non-M2-related mitochondrial 60 kDa protein found to be recognized by antimitochondrial antibody ( AMA) negative sera from patients with primary biliary cirrhosis ( PBC) has been shown to contain parts of the five F1- ATPase subunits α, β, γ, δ and ε. In this study, we examined whether this enzyme is, indeed, a target antigen in PBC. Methods Analysed were 60 AMA-positive/anti-M2-negative and 103 anti-M2-positive PBC patients, 46 patients with autoimmune hepatitis ( AIH), 35 patients with primary sclerosing cholangitis ( PSC), 110 patients with viral hepatitis, 40 patients with inflammatory bowel diseases ( IBD), 33 patients with connective tissue diseases (systemic lupus erythematosus, mixed connective tissue disease, Sjögren disease, systemic sclerosis) and 25 blood donors. The F1- ATPase-subunits α-δ were recombinantly expressed in E scherichia coli, purified and applied to ELISA and Western blotting. Results In all, 40 of the 60 AMA-positive/anti-M2-negative (67%) and 44 (43%) of the 103 anti-M2-positive PBC-sera reacted with at least one of the F1-subunits α-δ. The β- and γ-subunits were preferentially recognized. However, also up to 57% of patients with AIH and 34% of patients with PSC had anti-β- or γ-antibodies, while patients with viral hepatitis had these antibodies in up to 13%. Patients with IBD had anti-β and anti-γ-antibodies in up to 20 and 5% respectively. None of the patients with connective tissue diseases had antibodies to the β- and only 6% to the γ-subunit. Sera from healthy blood donors were negative. Conclusions Antibodies to the β- and γ-subunits of F1- ATPase are further AMAs in PBC but occur also in other autoimmune liver disorders; they may be, therefore, indicators for a general autoimmune process of the liver. [ABSTRACT FROM AUTHOR]