The main objective of the present program is to study the nature and significance of eye muscle (EM) antigens and the corresponding serum autoantibodies in patients with thyroid-associated ophthalmopathy (TAO), focusing on a thyroid and EM shared membrane antigen of 64 kDa which may explain the association of the eye disorder with Graves' hyperthyroidism and, less often, Hashimoto's thyroiditis. Because autoantibodies reactive with the 64 kDa antigen also cross-react with a shared antigen in other skeletal muscle, pancreas, ovary and testis a longer term objective of these studies must be to explain the absence of multiple tissue damage in patients with cross-reactive antibodies. One possibility is that the target cell surface antigens are not expressed at the time of exposure to cytotoxic antibodies. Such expression could be determined by environmental events such as infection, cytokines or thyroid hormones. For the performance of these studies the applicant would use immunological and molecular technology. Extensive gel electrophoresis studies would be carried out in which the relationship of the 64 kDa antigen to other EM membrane antigens of 55 and 95 kDa, the number of molecules at 64 kDa in the various target tissues, and the ability of patient autoantibodies to bind to the antigen on the surface of EM cells in vitro would be determined. Once a purified 64 kDa antigen has been obtained from 2D gel electrophoresis and nitrocellulose paper transfer, oligonucleotide probes can be synthesized and specific antisera raised for the screening of a thyroid cDNA expression library. cDNA inserts corresponding to the 64 kDa antigen would be sequenced and their amino acid sequences deduced. If the deduced amino acid sequences correspond to those of the original 2D gel - purified material one can be certain that the 64 kDa protein has been cloned. The cDNA inserts can be used as probes for the RNA message in the various tissues expressing the shared antigen. In the course of the studies the nature and significance of an EM specific antigen of 25 kDa would also be determined. If such an antigen is shown to be relevant to the pathogenesis of TAO it too would be cloned and the corresponding protein sequenced. TAO is a severe orbital inflammatory disorder which occasionally leads to blindness. An understanding of its immunopathogenesis, in particular the role of cytotoxic eye muscle antibodies in the initiation of the disease in patients with autoimmune thyroid disorders, would provide the basis for the early diagnosis and, ultimately, prevention of a disease which effects 1% of North American women.
