In vivo studies of ANCA induced glomerular inflammation

ANCA相关性肾小球损伤可能通过ANCA与中性粒细胞/单核细胞结合，导致中性粒细胞释放增多和内皮细胞损伤。我们的实验方法是基于ANCA是致病的，并负责介导最常见的全身性坏死性血管炎和新月体肾炎。第一个具体目标集中在ANCA诱导中性粒细胞/单核细胞活化的机制/S。拟议的体外和体内研究是互补的，将并行进行。体外研究将通过处理来自健康个体的细胞来检验抗体的F(ab‘)2部分与Fc部分的相对贡献。我们将设计反PR3和MPO F(ab‘)2类分子，带和不带FC区域来确定这一点。活体研究将调查疾病活动期、缓解期和复发期患者循环细胞的变化，即检测ANCA抗原的表面表达，以及与炎症相关的基因转录的变化。第二个特定目的是测试ANCA及其抗原PR3和MPO对内皮的影响。这些研究将与特定的目标1平行进行。我们知道PR3和MPO被转位到内皮细胞并诱导细胞凋亡，而MPO不能。我们将确定细胞进入的机制以及对导致细胞凋亡的细胞信号通路的影响。通过缺失分析，我们将确定PR3的结构域负责促凋亡活性。

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.