the hypothesis of this proposal is that autoantibodies (aab) present in Ro/SS-A (Ro) autoimmune sera are involved in the pathogenesis of LD-specific skin lesions such as SCLE and neonatal LE (NLE). Thus, characterization of the complete spectrum of aab specificities present in anti-Ro/SS-A sera is of critical importance to understanding the pathogenetic role played by the humoral autoimmune response that is seen in such patients. Previous studies have indicated that aab to native forms of calreticylin are produced in parallel to the anti-Ro/SS-A autoimmune response. In this application, the relationship that exists between these two patterns of autoimmunity will be examined, the significance of the anti-CR autoimmune response with respect to cutaneous LE will be explored, and the regulation of CR gene expression by UVB will be characterized. This will be accomplished using the following experimental approaches:
SPECIFIC AIM 1 : To Delineate the Relationship that Exists between CR and the conventional component of the Ro RNP. This will be accomplished by EMSA analysis employing CR mutants generated by the exonuclease recession technique, the SELEX technique, and the yeast two- hybrid protocol. The binding affinity of CR aab to recombinant CR-hYRNA complexes will be compared to that of recombinant CR alone.
SPECIFIC AIM 2 : to Determine the clinical and pathogenetic Significance of CR Aab in Cutaneous La patients. Various post-transnationally modified forms of rCr will be examined by ELISA and CIE for reactivity with sera from patients with cutaneous LE and disease controls. CR aab levels will be correlated with skin disease activity. The link between a-CR aab production and normal pregnancy will be further explored. The pathogenetic potential of affinity-purified anti-CR aab for triggering epidermal kerationocyte cytotoxicity will be explored.
SPECIFIC AIM 3 : To Examine the Effects of UV Radiation on the Genetic Regulation of CR. The CR gene response to UVB will be further characterized by reporter gene studies, competitive PCR, Northern blot analysis and nuclear run-on studies. The results of the studies proposed herein could provide further insights into the pathogenesis of anti-CR and anti-Ro aab-associated photosensitive Ld-specific skin diseases as well as explore the significance in cell biology of an intriguing new rheumatic disease autoantigen.
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