Graves' disease (GD) is an autoimmune disorder characterized by hyperthyroidism which is mediated by autoantibodies to the thyrotropin receptor (TSHr). Until now, it has not been possible to develop an animal model for GD due to the unavailability of purified TSHr protein. However, recently we expressed the extracellular domain of human TSHr protein (ETSHr) using a baculovirus expression system. The protein was purified to homogeneity and was shown to bind specifically both TSH and antibodies to TSHr. We used this protein to generate anti-ETSHr antibodies that can bind to native TSHr and block thyrotropin (TSH) binding. As a result of successful protein expression, we will now pursue our long-term objective, which is to establish an animal model and understand the pathogenic mechanics involved in the GD. In the present study we propose to use the recombinant ETSHr protein to induce experimental autoimmune Graves' disease (EAGD) in mice. Initial studies will be aimed at defining optimum conditions, such as age, sex, and genetic background of mice, and dose and route of inoculation of the antigen. Anti-TSHr antibody production will be monitored using an ELISA. Sera from seropositive animals will be tested for the presence of antibodies that can block TSH binding to TSHr (TB11) using a radioreceptor assay. Perturbation of thyroid function in TB11 positive animals will be detected by testing sera for both TSH and thyroxine levels using radioimmunoassays. Thyroid glands from mice with EAGD will be used for evaluating the pathology and for phenotyping infiltrating cells. Systematic characterization of T cell responses will be carried out using in vitro cell proliferation assays, by adoptive transfer experiments and by selective depletion of various subsets of T cells and B cells in vitro and in vivo. Finally, using a panel of overlapping synthetic peptides, that span the entire ETSHr, and peptide fragments of ETSHr we will attempt to map some of the B cell and T cell epitopes that are critical for EAGD. Together, these studies should result in the establishment of a mouse model for GD, that would allow systematic studies to understand regulation of immune response to TSHr. Moreover, these studies might lead to the identification of some of the T cell and B cell epitopes with potential implications for developing TSHr- specific immunotherapies.
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