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Autoantigen discovery is a critical challenge for the understanding and diagnosis of autoimmune diseases. While autoantibody markers in current clinical use have been identified through studies focused on individual disorders, we postulated that a reverse approach starting with a putative autoantigen to explore multiple disorders might hold promise. We here targeted the epidermal protein transglutaminase 1 (TGM1) as a member of a protein family prone to autoimmune attack. By screening sera from patients with various acquired skin disorders, we identified seropositive subjects with the blistering mucocutaneous disease paraneoplastic pemphigus. Validation in further subjects confirmed TGM1 autoantibodies as a 55% sensitive and 100% specific marker for paraneoplastic pemphigus. This gene-centric approach leverages the wealth of data available for human genes and may prove generally applicable for biomarker discovery in autoimmune diseases., Competing Interests: Competing interest statement: N.L., T.H., and O.K. are preparing to file a patent related to the use of transglutaminase 1 autoantibodies as a diagnostic marker., (Copyright © 2021 the Author(s). Published by PNAS.)

Narcolepsy type 1 (NT1) is a chronic sleep disorder caused by a specific loss of hypocretin-producing neurons. The incidence of NT1 increased in Sweden, Finland and Norway following Pandemrix®-vaccination, initiated to prevent the 2009 influenza pandemic. The pathogenesis of NT1 is poorly understood, and causal links to vaccination are yet to be clarified. The strong association with Human leukocyte antigen (HLA) DQB1*06:02 suggests an autoimmune pathogenesis, but proposed autoantigens remain controversial. We used a two-step approach to identify autoantigens in patients that acquired NT1 after Pandemrix®-vaccination. Using arrays of more than 9000 full-length human proteins, we screened the sera of 10 patients and 24 healthy subjects for autoantibodies. Identified candidate antigens were expressed in vitro to enable validation studies with radiobinding assays (RBA). The validation cohort included NT1 patients (n = 39), their first-degree relatives (FDR) (n = 66), population controls (n = 188), and disease controls representing multiple sclerosis (n = 100) and FDR to type 1 diabetes patients (n = 41). Reactivity towards previously suggested NT1 autoantigen candidates including Tribbles homolog 2, Prostaglandin D2 receptor, Hypocretin receptor 2 and α-MSH/proopiomelanocortin was not replicated in the protein array screen. By comparing case to control signals, three novel candidate autoantigens were identified in the protein array screen; LOC401464, PARP3 and FAM63B. However, the RBA did not confirm elevated reactivity towards either of these proteins. In summary, three putative autoantigens in NT1 were identified by protein array screening. Autoantibodies against these candidates could not be verified with independent methods. Further studies are warranted to identify hypothetical autoantigens related to the pathogenesis of Pandemrix®-induced NT1., (© 2020 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology.)

Dogs can spontaneously develop complex systemic autoimmune disorders, with similarities to human autoimmune disease. Autoantibodies directed at self-antigens are a key feature of these autoimmune diseases. Here we report the identification of interleukin enhancer-binding factors 2 and 3 (ILF2 and ILF3) as autoantigens in canine immune-mediated rheumatic disease. The ILF2 autoantibodies were discovered in a small, selected canine cohort through the use of human protein arrays; a method not previously described in dogs. Subsequently, ILF3 autoantibodies were also identified in the same cohort. The results were validated with an independent method in a larger cohort of dogs. ILF2 and ILF3 autoantibodies were found exclusively, and at a high frequency, in dogs that showed a speckled pattern of antinuclear antibodies on immunofluorescence. ILF2 and ILF3 autoantibodies were also found at low frequency in human patients with SLE and Sjögren's syndrome. These autoantibodies have the potential to be used as diagnostic biomarkers for canine, and possibly also human, autoimmune disease.

Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. High titer autoantibodies are a characteristic feature of APS1 and are often associated with particular disease manifestations. Pituitary deficits are reported in up to 7% of all APS1 patients, with immunoreactivity to pituitary tissue frequently reported. We aimed to isolate and identify specific pituitary autoantigens in patients with APS1. Immunoscreening of a pituitary cDNA expression library identified endothelin-converting enzyme (ECE)-2 as a potential candidate autoantigen. Immunoreactivity against ECE-2 was detected in 46% APS1 patient sera, with no immunoreactivity detectable in patients with other autoimmune disorders or healthy controls. Quantitative-PCR showed ECE-2 mRNA to be most abundantly expressed in the pancreas with high levels also in the pituitary and brain. In the pancreas ECE-2 was co-expressed with insulin or somatostatin, but not glucagon and was widely expressed in GH producing cells in the guinea pig pituitary. The correlation between immunoreactivity against ECE-2 and the major recognized clinical phenotypes of APS1 including hypopituitarism was not apparent. Our results identify ECE-2 as a specific autoantigen in APS1 with a restricted neuroendocrine distribution.

Autoimmune polyendocrine syndrome type 1 (APS1) is a monogenic disorder that features multiple autoimmune disease manifestations. It is caused by mutations in the Autoimmune regulator (AIRE) gene, which promote thymic display of thousands of peripheral tissue antigens in a process critical for establishing central immune tolerance. We here used proteome arrays to perform a comprehensive study of autoimmune targets in APS1. Interrogation of established autoantigens revealed highly reliable detection of autoantibodies, and by exploring the full panel of more than 9000 proteins we further identified MAGEB2 and PDILT as novel major autoantigens in APS1. Our proteome-wide assessment revealed a marked enrichment for tissue-specific immune targets, mirroring AIRE's selectiveness for this category of genes. Our findings also suggest that only a very limited portion of the proteome becomes targeted by the immune system in APS1, which contrasts the broad defect of thymic presentation associated with AIRE-deficiency and raises novel questions what other factors are needed for break of tolerance.

Autoimmune polyendocrine syndrome type 1 (APS1), a monogenic disorder caused by AIRE gene mutations, features multiple autoimmune disease components. Infertility is common in both males and females with APS1. Although female infertility can be explained by autoimmune ovarian failure, the mechanisms underlying male infertility have remained poorly understood. We performed a proteome-wide autoantibody screen in APS1 patient sera to assess the autoimmune response against the male reproductive organs. By screening human protein arrays with male and female patient sera and by selecting for gender-imbalanced autoantibody signals, we identified transglutaminase 4 (TGM4) as a male-specific autoantigen. Notably, TGM4 is a prostatic secretory molecule with critical role in male reproduction. TGM4 autoantibodies were detected in most of the adult male APS1 patients but were absent in all the young males. Consecutive serum samples further revealed that TGM4 autoantibodies first presented during pubertal age and subsequent to prostate maturation. We assessed the animal model for APS1, the Aire-deficient mouse, and found spontaneous development of TGM4 autoantibodies specifically in males. Aire-deficient mice failed to present TGM4 in the thymus, consistent with a defect in central tolerance for TGM4. In the mouse, we further link TGM4 immunity with a destructive prostatitis and compromised secretion of TGM4. Collectively, our findings in APS1 patients and Aire-deficient mice reveal prostate autoimmunity as a major manifestation of APS1 with potential role in male subfertility., (Copyright © 2015, American Association for the Advancement of Science.)

Context: NACHT leucine-rich-repeat protein 5 (NALP5)/maternal antigen that embryo requires (MATER) is an autoantigen in hypoparathyroidism associated with autoimmune polyendocrine syndrome type 1 (APS1) but is also expressed in the ovary. Mater is an autoantigen in experimental autoimmune oophoritis., Objectives: The objectives of the study were to determine the frequency of NALP5/MATER autoantibodies (NALP5/MATER-Ab) in women with premature ovarian insufficiency (POI) and in patients with autoimmune Addison's disease (AAD) and to evaluate whether inhibin chains are a target for autoantibodies in POI., Methods: Autoantibodies against NALP5/MATER and inhibin chains-α and -βA were determined by radiobinding assays in 172 patients with AAD without clinical signs of gonadal insufficiency, 41 women with both AAD and autoimmune POI [steroidogenic cell autoimmune POI (SCA-POI)], 119 women with idiopathic POI, 19 patients with APS1, and 211 healthy control subjects., Results: NALP5/MATER-Ab were detected in 11 of 19 (58%) sera from APS1 patients, 12 of 172 (7%) AAD sera, 5 of 41 (12%) SCA-POI sera, 0 of 119 idiopathic POI sera and 1 of 211 healthy control sera (P < .001). None of 160 POI sera, including 41 sera from women with SCA-POI and 119 women with idiopathic POI, and none of 211 healthy control sera were positive for inhibin chain-α/βA autoantibodies., Conclusions: NALP5/MATER-Ab are associated with hypoparathyroidism in APS1 but are present also in patients with AAD and in women with SCA-POI without hypoparathyroidism. Inhibin chains do not appear to be likely candidate targets of autoantibodies in human POI.

Patients with the autoimmune polyendocrine syndrome type I (APS-I), caused by mutations in the autoimmune regulator (AIRE) gene, and myasthenia gravis (MG) with thymoma, show intriguing but unexplained parallels. They include uncommon manifestations like autoimmune adrenal insufficiency (AI), hypoparathyroidism, and chronic mucocutaneous candidiasis plus autoantibodies neutralizing IL-17, IL-22, and type I IFNs. Thymopoiesis in the absence of AIRE is implicated in both syndromes. To test whether these parallels extend further, we screened 247 patients with MG, thymoma, or both for clinical features and organ-specific autoantibodies characteristic of APS-I patients, and we assayed 26 thymoma samples for transcripts for AIRE and 16 peripheral tissue-specific autoantigens (TSAgs) by quantitative PCR. We found APS-I-typical autoantibodies and clinical manifestations, including chronic mucocutaneous candidiasis, AI, and asplenia, respectively, in 49 of 121 (40%) and 10 of 121 (8%) thymoma patients, but clinical features seldom occurred together with the corresponding autoantibodies. Both were rare in other MG subgroups (n = 126). In 38 patients with APS-I, by contrast, we observed neither autoantibodies against muscle Ags nor any neuromuscular disorders. Whereas relative transcript levels for AIRE and 7 of 16 TSAgs showed the expected underexpression in thymomas, levels were increased for four of the five TSAgs most frequently targeted by these patients' autoantibodies. Therefore, the clinical and serologic parallels to APS-I in patients with thymomas are not explained purely by deficient TSAg transcription in these aberrant AIRE-deficient tumors. We therefore propose additional explanations for the unusual autoimmune biases they provoke. Thymoma patients should be monitored for potentially life-threatening APS-I manifestations such as AI and hypoparathyroidism., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Interstitial lung disease (ILD) is a complex and heterogeneous disorder that is often associated with autoimmune syndromes. Despite the connection between ILD and autoimmunity, it remains unclear whether ILD can develop from an autoimmune response that specifically targets the lung parenchyma. We examined a severe form of autoimmune disease, autoimmune polyglandular syndrome type 1 (APS1), and established a strong link between an autoimmune response to the lung-specific protein BPIFB1 (bactericidal/permeability-increasing fold-containing B1) and clinical ILD. Screening of a large cohort of APS1 patients revealed autoantibodies to BPIFB1 in 9.6% of APS1 subjects overall and in 100% of APS1 subjects with ILD. Further investigation of ILD outside the APS1 disorder revealed BPIFB1 autoantibodies present in 14.6% of patients with connective tissue disease-associated ILD and in 12.0% of patients with idiopathic ILD. The animal model for APS1, Aire⁻/⁻ mice, harbors autoantibodies to a similar lung antigen (BPIFB9); these autoantibodies are a marker for ILD. We found that a defect in thymic tolerance was responsible for the production of BPIFB9 autoantibodies and the development of ILD. We also found that immunoreactivity targeting BPIFB1 independent of a defect in Aire also led to ILD, consistent with our discovery of BPIFB1 autoantibodies in non-APS1 patients. Overall, our results demonstrate that autoimmunity targeting the lung-specific antigen BPIFB1 may contribute to the pathogenesis of ILD in patients with APS1 and in subsets of patients with non-APS1 ILD, demonstrating the role of lung-specific autoimmunity in the genesis of ILD.

Background: Lymphocytic hypophysitis is an organ-specific autoimmune disease of the pituitary gland. A specific and sensitive serological test currently does not exist to aid in the diagnosis., Objective: To identify target autoantigens in lymphocytic hypophysitis and develop a diagnostic assay for these proteins., Design/methods: A pituitary cDNA expression library was immunoscreened using sera from four patients with lymphocytic hypophysitis. Relevant cDNA clones from screening, along with previously identified autoantigens pituitary gland-specific factor 1a and 2 (PGSF1a and PGSF2) and neuron-specific enolase (NSE) were tested in an in vitro transcription and translation immunoprecipitation assay. The corticotroph-specific transcription factor, TPIT, was investigated separately as a candidate autoantigen., Results: Significantly positive autoantibody reactivity against TPIT was found in 9/86 hypophysitis patients vs 1/90 controls (P = 0.018). The reactivity against TPIT was not specific for lymphocytic hypophysitis with autoantibodies detectable in the sera from patients with other autoimmune endocrine diseases. Autoantibodies were also detected against chromodomain-helicase-DNA binding protein 8, presynaptic cytomatrix protein (piccolo), Ca(2+)-dependent secretion activator, PGSF2 and NSE in serum samples from patients with lymphocytic hypophysitis, but at a frequency that did not differ from healthy controls. Importantly, 8/86 patients with lymphocytic hypophysitis had autoantibodies against any two autoantigens in comparison with 0/90 controls (P = 0.0093)., Conclusions: TPIT, a corticotroph-specific transcription factor, was identified as a target autoantigen in 10.5% of patients with lymphocytic hypophysitis. Further autoantigens related to vesicle processing were also identified as potential autoantigens with different immunoreactivity patterns in patients and controls.

Patients with autoimmune polyendocrine syndrome type 1 (APS-1) suffer from multiple organ-specific autoimmunity with autoantibodies against target tissue-specific autoantigens. Endocrine and nonendocrine organs such as skin, hair follicles, and liver are targeted by the immune system. Despite sporadic observations of pulmonary symptoms among APS-1 patients, an autoimmune mechanism for pulmonary involvement has not been elucidated. We report here on a subset of APS-1 patients with respiratory symptoms. Eight patients with pulmonary involvement were identified. Severe airway obstruction was found in 4 patients, leading to death in 2. Immunoscreening of a cDNA library using serum samples from a patient with APS-1 and obstructive respiratory symptoms identified a putative potassium channel regulator (KCNRG) as a pulmonary autoantigen. Reactivity to recombinant KCNRG was assessed in 110 APS-1 patients by using immunoprecipitation. Autoantibodies to KCNRG were present in 7 of the 8 patients with respiratory symptoms, but in only 1 of 102 APS-1 patients without respiratory symptoms. Expression of KCNRG messenger RNA and protein was found to be predominantly restricted to the epithelial cells of terminal bronchioles. Autoantibodies to KCNRG, a protein mainly expressed in bronchial epithelium, are strongly associated with pulmonary involvement in APS-1. These findings may facilitate the recognition, diagnosis, characterization, and understanding of the pulmonary manifestations of APS-1.

Background: Autoimmune polyendocrine syndrome type 1 (APS-1) is a multiorgan autoimmune disorder caused by mutations in AIRE, the autoimmune regulator gene. Though recent studies concerning AIRE deficiency have begun to elucidate the molecular pathogenesis of organ-specific autoimmunity in patients with APS-1, the autoantigen responsible for hypoparathyroidism, a hallmark of APS-1 and its most common autoimmune endocrinopathy, has not yet been identified., Methods: We performed immunoscreening of a human parathyroid complementary DNA library, using serum samples from patients with APS-1 and hypoparathyroidism, to identify patients with reactivity to the NACHT leucine-rich-repeat protein 5 (NALP5). Subsequently, serum samples from 87 patients with APS-1 and 293 controls, including patients with other autoimmune disorders, were used to determine the frequency and specificity of autoantibodies against NALP5. In addition, the expression of NALP5 was investigated in various tissues., Results: NALP5-specific autoantibodies were detected in 49% of the patients with APS-1 and hypoparathyroidism but were absent in all patients with APS-1 but without hypoparathyroidism, in all patients with other autoimmune endocrine disorders, and in all healthy controls. NALP5 was predominantly expressed in the cytoplasm of parathyroid chief cells., Conclusions: NALP5 appears to be a tissue-specific autoantigen involved in hypoparathyroidism in patients with APS-1. Autoantibodies against NALP5 appear to be highly specific and may be diagnostic for this prominent component of APS-1., (Copyright 2008 Massachusetts Medical Society.)

Patients with autoimmune polyendocrine syndrome type 1 often have autoantibodies against neurotransmitter synthesizing enzymes, including the pyridoxal phosphate-dependent enzymes glutamic acid decarboxylase and aromatic L-amino acid decarboxylase. Using a candidate approach, we have identified the histamine-synthesizing enzyme histidine decarboxylase, also pyridoxal phosphate dependent, as an autoantigen in this disorder. Anti-histidine decarboxylase antibodies reacting with in vitro translated antigen were found in 36/97 (37%) of autoimmune polyendocrine syndrome type 1 patients studied. The antibodies also reacted with the native enzyme in HMC-1 cell lysates and did not cross-react with the highly homologous aromatic L-amino acid decarboxylase. Anti-histidine decarboxylase antibodies were associated with a history of intestinal dysfunction (P = 0.017). Gastric and duodenal biopsies from a patient with anti-histidine decarboxylase antibodies were studied by immunohistochemistry. The oxyntic mucosa was found to lack the histamine producing enterochromaffin-like cells, suggestive of an autoimmune destruction. To our knowledge, this is the first report of autoantibodies against histidine decarboxylase and absence of gastric enterochromaffin-like cells.

To identify candidate autoantigens associated with arthritis, a rat chondrocyte cDNA library was immunoscreened with serum from a patient with rheumatoid arthritis. One isolated cDNA encoded part of AHNAK, a 700-kDa phosphoprotein with DNA binding properties, that appears to be involved in several signal transduction pathways. Immunoreactivity against an in vitro translated human AHNAK fragment was detected in 4.6% (5/109) of patients with rheumatoid arthritis, 29.5% (18/61) of patients with systemic lupus erythematosus (SLE), and 1.2% (2/172) of blood donors. Anti-AHNAK antibodies reacted with a recombinant human AHNAK fragment and with native AHNAK from C32 cell lysates. In vitro translated AHNAK fragment could be cleaved by granzyme B and caspase-3. Anti-AHNAK positive SLE patients had a higher frequency of homogeneous antinuclear antibody staining patterns and a lower frequency of recent mucosal ulcerations. This is the first report that AHNAK can be targeted by the immune system in autoimmune disease.

Vitiligo is common in the hereditary disorder autoimmune polyendocrine syndrome type I (APS I). Patients with APS I are known to have high titer autoantibodies directed against various tissue-specific antigens. Using sera from APS I patients for immunoscreening of a cDNA library from human scalp, we identified the transcription factors SOX9 and SOX10 as novel autoantigens related to this syndrome. Immunoreactivity against SOX9 was found in 14 (15%) and against SOX10 in 20 (22%) of the 91 APS I sera studied. All patients reacting with SOX9 displayed reactivity against SOX10, suggesting shared epitopes. Among the 19 patients with vitiligo, 12 (63%) were positive for SOX10 (p < 0.0001). Furthermore, three of 93 sera from patients with vitiligo unrelated to APS I showed strong reactivity against SOX10, which may indicate a more general role of SOX10 as an autoantigen in vitiligo.

Patients with the autosomal recessively inherited autoimmune polyendocrine syndrome type I (APS I) have autoantibodies directed against several endocrine and nonendocrine organs. In this study a new autoantigen related to this syndrome, tyrosine hydroxylase, was identified in sera from patients with alopecia areata through immunoscreening of a scalp cDNA library. Immunoreactivity against in vitro expressed tyrosine hydroxylase was found in 41 (44%) of the 94 APS I patients studied and this reactivity correlated with the presence of alopecia areata (P = 0.02). These findings further stress the importance of enzymes involved in neurotransmitter biosynthesis as important immune targets in APS I., (Copyright 2000 Academic Press.)

Background: Autoimmune polyendocrine syndrome type 1 (APS1) is an autosomal recessive disorder with both endocrine and non-endocrine features. Periodic gastrointestinal dysfunction occurs in 25-30% of APS1 patients. We aimed to identify an intestinal autoantigen., Methods: A human duodenal cDNA library was immunoscreened with serum samples from APS1 patients. A positive clone was identified and used for in-vitro transcription and translation, followed by immunoprecipitation with serum samples from 80 APS1 patients from Norway, Finland, and Sweden. Sections of normal and APS1-affected small intestine were immunostained with serum from APS1 patients and specific antibodies. An enzyme-inhibition assay was used to characterise the autoantibodies., Findings: We isolated a cDNA clone coding for tryptophan hydroxylase. 48% (38/80) of APS1 patients had antibodies to tryptophan hydroxylase, whereas no reactivity to this antigen was detected in patients with other autoimmune diseases (n=372) or healthy blood donors (n=70). 89% (17/19) of APS1 patients with gastrointestinal dysfunction were positive for antibodies to tryptophan hydroxylase, compared with 34% (21/61) of patients with no gastrointestinal dysfunction (p<0.0001). Serum from antibody-positive APS1 patients specifically immunostained tryptophan-hydroxylase-containing enterochromaffin cells in normal duodenal mucosa. No serotonin-containing cells were seen in duodenal biopsy samples from APS1 patients. Serum from antibody-positive APS1 patients almost completely inhibited activity of tryptophan hydroxylase., Interpretation: Tryptophan hydroxylase is an endogenous intestinal autoantigen in APS1, and there is an association between antibodies to the antigen and gastrointestinal dysfunction. Analysis of antibodies to tryptophan hydroxylase may be a valuable diagnostic tool to predict and monitor gastrointestinal dysfunction in APS1.

Autoimmune chronic active hepatitis (AI-CAH) is a feared component of autoimmune polyendocrine syndrome type I (APS I). In this study, immunoreactivity was assessed in sera from eight APS I patients, of whom three had AI-CAH, in an attempt to identify hepatic autoantigens. We performed indirect immunofluorescence staining of human and rat liver sections, Western blots on subcellular fractions of human and rat liver, immunoprecipitations of labelled aromatic L-amino acid decarboxylase (AADC) and cytochrome P450IA2 (CYP IA2) expressed by an in vitro transcription and translation system and studies of enzymatic activity. Autoantibodies against AADC were present in sera from all eight APS I patients, while immunoreactivity against CYP IA2 was only found in sera from the three APS I patients with AI-CAH. Enzymatic activity of CYP IA2 was inhibited by sera from APS I patients with AI-CAH but not by control sera. Our results show that CYP IA2 and AADC constitute hepatic autoantigens in patients with APS I and that immunoreactivity against CYP IA2 is associated with the presence of AI-CAH.

Different autoantigens are thought to be involved in the pathogenesis of insulin-dependent diabetes mellitus, and they may account for the variation in the clinical presentation of the disease. Sera from patients with autoimmune polyendocrine syndrome type I contain autoantibodies against the beta-cell proteins glutamate decarboxylase and an unrelated 51-kDa antigen. By screening of an expression library derived from rat insulinoma cells, we have identified the 51-kDa protein as aromatic-L-amino-acid decarboxylase (EC 4.1.1.28). In addition to the previously published full-length cDNA, forms coding for a truncated and an alternatively spliced version were identified. Aromatic L-amino acid decarboxylase catalyzes the decarboxylation of L-5-hydroxytryptophan to serotonin and that of L-3,4-dihydroxyphenylalanine to dopamine. Interestingly, pyridoxal phosphate is the cofactor of both aromatic L-amino acid decarboxylase and glutamate decarboxylase. The biological significance of the neurotransmitters produced by the two enzymes in the beta cells remains largely unknown.

Autoimmune adrenal insufficiency is associated with premature ovarian failure at frequencies of 10-20%. In these patients a reactivity against an unknown steroid cell antigen in both the adrenal glands and gonads has been described. We have recently identified the cytochrome P450 enzyme 21-hydroxylase and the side-chain cleavage enzyme (SCC) as the major adrenal autoantigens in Addison's disease and the rare autoimmune polyendocrine syndrome type I (APS-I), respectively. In an attempt to identify the steroid cell antigen, sera from patients with Addison's disease (n = 13) and APS-I (n = 7) that reacted with Leydig cells were selected. Preparations of isolated human granulosa cells, isolated rat Leydig cells, and fractions of human placenta were used in Western blots. All sera were also tested against bacterially expressed 21-hydroxylase, SCC, and 17 alpha-hydroxylase. The SCC was recognized by sera from patients with Addison's disease and those with APS-I. In addition, a majority of the sera (n = 12) reacted with a novel 51-kilodalton autoantigen present in granulosa cells and placenta. The results show that the steroid cell autoantigen consists mainly of the SCC and a novel protein of unknown function.

Beta-cell function and islet cell antibodies were studied in six patients with autoimmune polyendocrine syndrome type I. All suffered from mucocutaneous candidiasis, five had adrenocortical insufficiency and three hypoparathyroidism. All sera contained high titres of antibodies staining islets of Langerhans. Reactivity against glutamate decarboxylase, predominantly the 65 kDa isoform, was detected by immunoprecipitations and Western blots in five of the six sera, and all six sera immunoprecipitated a 51 kDa antigen from [35S]-methionine labelled rat islet cell lysates. No reactivity against this latter antigen was found in sera of patients with Type 1 (insulin-dependent) diabetes mellitus (n = 9), Graves' disease (n = 5), autoimmune gastritis (n = 4), idiopathic Addison's disease (n = 7), or stiff-man syndrome (n = 2). The 51 kDa antigen was also detected by Western blots using homogenates of rat islets and autoimmune polyendocrine syndrome type I patient sera, whereas no such reactivity was found with homogenates of testes, adrenals, small intestine, spleen, exocrine pancreas or brain. Moreover, the 51 kDa antigen was present in the rat insulinoma cell line RINm 5F but not in the SV-40 transformed, monkey kidney cell line COS, when examined by immunoprecipitations of [35S]-methionine labelled cell lysates and by Western blots. None of the patients with autoimmune polyendocrine syndrome type I had symptoms of diabetes and their insulin responses to glucose challenge were normal. The data illustrate that patients with autoimmune polyendocrine syndrome type I present an autoimmune response against islets of Langerhans, which is apparently different from that associated with classic Type 1 diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmune polyendocrine syndrome type I (APS I) and idiopathic Addison's disease are both disorders with adrenal insufficiency but with differences in genetic background, clinical presentation, and extent of extraadrenal manifestations. In this study the major adrenal autoantigen identified with sera from patients with APS I was characterized by analyses using indirect immunofluorescence, Western blots of adrenal subcellular fractions and of recombinant proteins, immunoprecipitations of [35S]methionine-labeled lysates of a human steroid-producing cell line, and studies of enzymatic activity. Sera from patients with APS I, identifying cells in adrenal glands and testes involved in steroid synthesis, reacted in Western blots with a 53-kD antigen, which comigrated with the cytochrome P450 cholesterol side chain cleavage enzyme (SCC). The sera also immunoprecipitated this protein from lysates of radiolabeled adrenal cells. The enzymatic activity of SCC was inhibited by the APS I sera but not by control sera. Sera from patients with idiopathic Addison's disease did not react with the SCC. The results show that the autoimmune responses towards adrenal tissue in patients suffering from APS I and Addison's disease are remarkably selective and suggest that a determination of the antigen involved in a patient with autoimmune adrenal insufficiency will have diagnostic as well as prognostic implications.

Sera of patients with Addison's disease contain autoantibodies recognizing antigen(s) in the adrenal cortex. In the present study we have examined the antigen expression in normal (n = 6) and pathological human adrenal tissues (n = 24) and also in the human steroid-producing adrenocortical cell line NCI-H295. Sera from two patients with Addison's disease were selected as they strongly stained the human adrenal gland and identified a 54 kDa autoantigen previously demonstrated as 21-hydroxylase. These sera reacted with normal human adrenal cortex (n = 6), all hyperplasias (n = 5) and all the adrenocortical cancers (n = 9), whereas slight or no reactivity was observed in the adenomas without any detectable excess of peripheral steroids (n = 4). Both patient sera reacted in an identical manner with each tissue specimen and they also reacted strongly with the steroid-producing cell line. The data demonstrate that the expression of the Addisonian autoantigen correlates with the functional activity of adrenocortical neoplasms. Furthermore they suggest, that immunohistochemical stainings for steroid-producing enzymes may be clinically useful in the characterization of adrenal lesions.

Recent data suggest that the enzyme glutamic acid decarboxylase (GAD) may be a primary beta-cell antigen involved in the autoimmune response leading to insulin dependent diabetes mellitus (IDDM). Following three days culture of human islets at different glucose concentrations (5.6, 11 or 28 mM), there was a single 65 kDa/GAD band in the islet extracts, as assessed by immunoprecipitation both with serum from a newly diagnosed IDDM patient and a sheep anti-GAD serum. The synthesis of GAD was strongly stimulated at the higher glucose concentrations. Likewise, Western blot analysis indicated a glucose-induced increase of GAD in the islets. The data suggest that an enhanced function of human islet cells increases expression of GAD65. Possibly, this could exacerbate the autoimmune assault in the early stages of IDDM, and be of practical importance in attempts to ameliorate the autoimmune response towards the beta-cells in IDDM.

Sera from patients with idiopathic Addison's disease commonly react with the zona glomerulosa of adrenal cortex. We used high-resolution western blot analysis of an adrenal microsomal fraction to investigate the target of these antibodies. A protein with an apparent molecular weight of 54 kDa was recognised as the common and major component. Sera identifying this autoantigen (from 12 of 16 patients) also showed strong immunofluorescence staining of a steroid-producing human adrenal adrenocortical cell line, NCI-H295. On application of antisera specific for different cytochrome P450 steroidogenic enzymes (side-chain cleavage enzyme, 21-hydroxylase, 17 alpha-hydroxylase, 11 beta-hydroxylase) the mobility of the 54 kDa protein in western blots corresponded to that of 21-hydroxylase. This parallel behaviour was confirmed by immunoprecipitation, electrophoresis, and autoradiography with the various sera and 35S-methionine-labelled NCI-H295 cell lysates. Preabsorptions of 35S-methionine-labelled cell lysates with the antiserum to 21-hydroxylase, but not with the other enzyme antisera, abolished precipitation of the 54 kDa autoantigen with the patient sera. These results indicate that 21-hydroxylase (P450c21), prominent in the zona glomerulosa of the adrenal cortex, is a major autoantigen in idiopathic Addison's disease.

This study examined the relationship between insulin secretion and expression of the 64 kDa/glutamic acid decarboxylase autoantigen in pancreatic islets. Islets isolated from Wistar rats were cultured for 3 days under different conditions: in 5.5 mmol/l glucose with or without alpha-ketoisocaproic acid or glipizide and in 28 mmol/l glucose with or without diazoxide. The 64 kDa/glutamic acid decarboxylase autoantigen was precipitated from lysates of [35S]-methionine-labelled islets with sera from patients with Type 1 (insulin-dependent) diabetes mellitus and identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. In parallel, insulin contents of the islets and the media were determined as well as the rates of glucose-stimulated (pro)insulin biosynthesis. alpha-Ketoisocaproic acid and glipizide were found to stimulate the expression of the 64 kDa/glutamic acid decarboxylase autoantigen and also the rate of insulin secretion. Diazoxide on the other hand reduced the rate of the 64 kDa/glutamic acid decarboxylase autoantigen synthesis in parallel with an inhibition of glucose-stimulated insulin release. Under most of the conditions employed, (pro)insulin biosynthesis was not affected. The correlation found between the rate of insulin release and expression of the 64 kDa/glutamic acid decarboxylase autoantigen might provide an explanation for the earlier observed relationship between the functional demands on the Beta cells and their rate of destruction which may result in diabetes.

This study examined the effect of various extracellular glucose concentrations on the expression of a previously described 64,000-Mr islet cell autoantigen associated with insulin-dependent diabetes mellitus. The protein was precipitated from patient serums incubated with Triton X-100 lysates of [35S]methionine-labeled rat pancreatic islets that had been cultured in 5, 11, or 28 mM glucose for 6 h or 3 days. In both types of experiment, 28 mM glucose was the most efficient stimulator of 64,000-Mr autoantigen production. In contrast, the class I antigens of the major histocompatibility complex, precipitated by a rabbit polyclonal antiserum, were not influenced by differences in glucose concentrations. Our data indicate that expression of islet cell antigens may be increased during the course of hyperglycemia and suggest that the functional activity of islet cells influences their antigenicity.