ANCA-associated glomerular injury may be mediated by ANCA binding to neutrophils/monocytes, resulting in enhanced neutrophil granule release and endothelial cell damage. Our experimental approach is based on the premise that ANCA are pathogenic and are responsible for mediating the most common forms of systemic necrotizing vasculitis and crescentic glomerulonephritis. The first Specific Aim focuses on the mechanism/s of ANCA-induced neutrophil/monocyte activation. The proposed in vitro and in vivo studies are complementary and will be done in parallel. The in vitro studies will examine the relative contribution of the F(ab')2 portion of the antibody versus the Fc portion, by treating cells from healthy individuals. We will engineer anti-PR3 and MPO F(ab')2- like molecules, with and without Fc regions to determine this. The in vivo studies will investigate changes in circulating cells from patients with active disease, in remission and at relapse, i.e., examine the surface expression ANCA antigens, and examine the changes in transcription of genes associated with inflammation. The second Specific Aim will test the effects of ANCA and its antigens, PR3 and MPO, on the endothelium. These studies will be done in parallels with Specific Aim 1. We known that PR3 and MPO are translocated into endothelial cells and PR3 induced apoptosis but MPO did not. We will determine the mechanism of cell entry and the effects on cell signaling pathways that lead to apoptosis. Be deletion analysis we will determine the domain of PR3 responsible for the pro-apoptotic activity.
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