The applicant, and her mentor, John Harley, M.D., Ph.D., have recently demonstrated that normal rabbits immunized with specific short peptides derived from the Sm autoantigen develop high levels of both anti-Sm and anti-nRNP antibodies; the peptides used as immunogens were selected since they are also bound by human lupus autoantibodies. The peptide-immunized rabbits, in addition to anti-Sm and anti-nRNP antibodies, also develop positive ANA, antibodies to DNA, and clinical features reminiscent of human lupus including proteinuria, red cell casts, renal insufficiency, hypoalbuminemia, thrombocytopenia, alopecia, and seizures. The antibodies that develop to Sm and nRNP are apparently of high titer in that they precipitate in double immunodiffusion assays and bind RNP antigens in solution phase and by immunoblots. In essence, by immunizing rabbits with peptides, Dr. James and Dr. Harley have induced a syndrome in rabbits that resembles human lupus. In more recent, preliminary studies that form the foundation of the present proposal, Dr. James has shown that certain strains of inbred mice also respond to Sm peptide immunization with epitope spreading and genesis of autoantibodies to other parts of the Sm and nRNP autoantigens, as well as positive ANA and antibodies to DNA. In comparison, certain other strains respond to peptide immunization with antibodies against the immunogenetic peptide, but do not develop epitope spreading and other humoral features of lupus. Dr. James now proposes in the current project to identify the genes involved in this peptide-induced lupus model, using the technique of recombinant inbred (RI) mice strains. Human homologies to murine genes will then be sought.
Five specific aims are proposed. First, progenitor strains of recombinant inbred mice which do and do not develop peptide induced autoimmunity will be ascertained. The essence of this aim is to carefully identify strains of mice which do and do not develop lupus autoimmunity after immunization, with lupus autoimmunity being defined as spreading of the humoral immune response beyond the peptide of immunization to other regions of the Sm and nRNP autoantigens, as well as the development of positive ANA and anti-DNA antibodies as determined by Crithidiae immunofluorescence. These experiments are already underway with eleven different RI progenitor strains. Preliminary results from these studies suggest that both responder and non-responder strains have been identified. Second, recombinant inbred substrains, produced from a cross between a progenitor responder and non-responder, will be analyzed for evidence of peptide-induced lupus. In these experiments, the RI set derived from the progenitor strains selected in Aim 1 will be immunized with an Sm (spliceosomal) peptide and analyzed along with control mice. Animals will be assessed for development of lupus autoimmunity as defined in Aim 1. In the third specific aim, the murine locus (or loci) associated with peptide-induced lupus will be defined. In these experiments, RI sets will be analyzed for linkage of known markers of the affected progenitor strain that has evidence of peptide-induced lupus autoimmunity and/or clinical symptoms. These analyses will be accomplished using a computer program, MapManager V.2.5. Linkage will be determined by Chi square analysis using confidence intervals of p<0.01. Fourth, the identity of any loci found in specific aim four will be confirmed by classical genetic techniques and potential candidate genes examined. In these studies, confirmation of linkage will be obtained either using congenic lines (if one gene is involved in the production of peptide-induced autoimmunity) or via traditional back-cross analyses using the original progenitor strains of the RI set (if multiple loci are involved). In the latter case, gene mapping will be done using conventional markers as well as dispersed polymorphic DNA markers available from the mouse genome project. Finally, in aim 5, human genes homologous to those related to peptide induced autoimmunity in mice will be sought.
This aim depends upon the mapping of loci involved in peptide-induced lupus autoimmunity and establishment of candidate genes, as planned in specific aims 3 and 4. If such genes are found, then the comparative human-mouse map in which 80% of the mouse genome is accounted for, will be used to establish probable location of human homologous genes.
This specific aim will depend upon a large genetic linkage study in human SLE currently being performed by Dr. Harley, and which already contains 30 pedigrees with a second cohort of an additional 30 pedigrees planned.
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