SPONTANEOUS DIABETES--PATHOGENESIS AND TREATMENT

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Destruction of beta cells in diabetes is thought to be caused by a failure of self-tolerance whereby autoreactive T cells fail to undergo negative selection during intrathymic maturation. We have found that implantation of a small number of beta cells into the thymus of neonatal diabetes-prone BB rats prevents the expected autoimmune destruction of islets in the native pancreas. This provides us with an ideal model for dissection of the islet complex to define the specific autoantigenic component(s) of islets which are the targets of diabetes and which therefore could also be useful in immunotherapy. Cellular components of islets (endocrine cells vs antigen presenting cells) and subcellular elements (glutamic acid decarboxylase and insulin) will be evaluated as the likely diabetogenic autoantigens. We will inoculate the thymus of neonatal BB rats with whole islets, APC free islets, GAD protein, insulin, and non-islet cells which express either GAD (neuronal cells) or insulin (pituitary cells genetically engineered to produce insulin). Recipients will be monitored to determine whether any of these treatments, like intrathymic inoculation of whole islets, has a protective influence on the development of diabetes. A surprising finding made in our preliminary immunohistochemical search of the thymus for transplanted neuronal cells which express GAD was that thymic cells themselves also express GAD. The additional observation that the anatomical pattern of intrathymic expression of GAD differs in BB rats and normal rats is intriguing and could have etiological implications. With molecular assays we will confirm the presence of thymic GAD. We will study GAD expression in the thymus of other normal and diabetes-prone animals (e.g. NOD mice) to determine the generality of the observation. We will also study whether the thymic pattern of GAD expression can be normalized by maneuvers known to prevent diabetes in BB rats, e.g. cross breeding with normal rats, neonatal transplantation of bone marrow from normal donors. t is somewhat difficult to reconcile the crucial importance of GAD as the key diabetogenic autoantigen with its presence (rather than expected absence) from the thymus of diabetes prone rats, unless faulty intrathymic presentation of this autoantigen in BB rats prevents their ability to negatively select beta cell autoreactive T cells. This possibility will be explored by inoculating normal antigen-presenting cells into the thymus of BB rats. Since we can prevent diabetes by thymic manipulation of neonatal BB rats, we will also explore the use of these strategies in animals with acute onset diabetes to determine whether islets under autoimmune attack can be rescued. Similar methods will also be employed in older animals with established diabetes since eventual human application of these methods could probably not be employed in newborns. The latter studies will include novel methods of preventing allogeneic rejection and/or autoimmune destruction of transplanted islets with the use of a cyclic CD4 peptide analogue.