Humoral immunity to recombinant human autoantigens in organ-specific autoimmune disease

Sammanfattning: HUMORAL IMMUNITY TO RECOMBINANT HUMAN AUTOANTIGENS IN ORGAN-SPECIFIC AUTOIMMUNE DISEASESAlberto Falorni, from Endocrine & Diabetes Unit, Department of Molecular Medicine, KarolinskaInstitute, Stockholm, Sweden Insulin-dependent diabetes mellitus (IDDM) and idiopathic Addison's disease are organ-specificautoimmune diseases characterized by the presence of circulating autoantibodies. The role ofautoantibodies in the pathogenesis of these diseases is not fully understood, but their presence can beused as a marker for diagnosis and prediction. The development of novel autoantibody assays is acritical step in improving both the accuracy of IDDM prediction and our understanding of themechanims of autoantibody formation. Several islet proteins are target of circulating autoantibodies inIDDM, and the enzyme glutamic acid decarboxylase (GAD) is a major autoantigen in this disease. In our project we developed sensitive and specific radiobinding assays for GAD65Ab andGAD67Ab, which used in vitro translated recombinant human GAD65 or rat GAD67. Subsequentlywe improved the critical steps of our GAD65Ab assay, and we developed a novel, semi-automatedassay which allows a single operator to analyse up to 400 serum samples per week. Our GAD65Abassay was validated in international workshops, and found to have highest diagnostic sensitivity andspecificity for IDDM. Using our GADAb immunoassays, we have demonstrated the high occurrence ofGAD65Ab in Japanese patients with short-duration IDDM. GAD65Ab were also found in 10/20 (50%)slowly progressive IDDM patients, that demonstrates an underlying autoimmunity in these patients. InCaucasians, GAD65Ab were found in 75-76% (and GAD67Ab in 15-20%) of IDDM patients and only1-1.5% of healthy subjects. Occurrence of GAD65Ab was both age- and gender-dependent, andprevalence of GAD65Ab resulted higher in female and in adult patients. In IDDM patients with clinicalonset between age 20 and 40 years, presence of GAD65Ab had the highest diagnostic sensitivity for thedisease, as compared to ICA or IAA. GAD65Ab, ICA and IAA were found in 11.5%, 8.1%-and10.8%, respectively, of offspring of IDDM father and in only 2.1%, 1.4% and 2.8%, respectively, ofoffspring of IDDM mothers. These data were interpreted to demonstrate that IDDM mothers transmitislet autoimmunity less frequently to their offspring than IDDM fathers. As the risk for IDDM isapproximately 5-fold lower in offspring of IDDM mothers than offspring of IDDM fathers, the resultsof our study demonstrate that prevalence of autoantibodies in offspring of IDDM parents correlates withthe disease risk. In two studies, GAD65Ab and GAD67Ab were found in Graves' or APS I patients,also in absence of clinical signs of IDDM. We used hybrid molecules generated by substitution ofregions of GAD65 with homologous regions of GAD67 to localise the major, IDDM-relatedGAD65Ab epitopes in the central and carboxy-terminal domains of GAD65. In the same study, titres ofcarboxy-terminal GAD65 epitope-specific antibodies were significantly higher in IDDM patients than inhealthy controls and may, hypothetically, be used to distinguish IDDM from healthy children. The enzyme steroid-21-hydroxylase (P450c21) is a major autoantigen in autoimmune Addison'sdisease. To determine the diagnostic sensitivity and specificity of P450c21Ab for autoimmuneAddison's disease we developed a novel radiobinding assay using in vitro translated P450c21.P450c21Ab were found in 16/16 (100%) idiopathic Addison patients with less than 20 years diseaseduration, and in 8/12 (67%) patients with more than 20 years disease duration. Levels of P450c21Abinversely correlated with disease duration. The diagnostic sensitivity of P450c21Ab, as detected in ourradiobinding assay, was compared to that of a classical indirect immunofluorescence assay for adrenalcortex autoantibodies, and found to be higher. In two separate studies, P450c21Ab were not found in 5patients with adrenoleukodystrophy and in 7 patients with either post-tuberculosis or post-adrenalectomy Addison's disease. In patients with other endocrine autoimmune diseases, P450c21Abwere shown to be highly specific for Addison's disease. We have also shown that single, naturally-occuring amino acid substitutions of the COOH-terminal domain of P450c21 inhibit antibody binding,by modifying one or more conformation-dependent autoantibody epitope(s). In conclusion, we have demonstrated that presence of GAD65Ab or P450c21Ab is stronglyassociated with IDDM or Addison's disease, respectively. Our GAD65Ab and P450c21Ab assaysshould prove useful to identify subjects at high risk for these organ-specific autoimmune diseases.Key words: IDDM, Addison's disease, autoantibodies, recombinant autoantigens, radioimmunoassay, disease prediction, glutamic-acid decarboxylase, steroid-21-hydroxylaseISBN-91-628-2121-0