In vivo studies of ANCA induced glomerular inflammation

ANCA相关的肾小球损伤可能通过ANCA与中性粒细胞/单核细胞结合介导，导致中性粒细胞颗粒释放增强和内皮细胞损伤。我们的实验方法是基于这样的前提，即ANCA是致病性的，并负责介导最常见形式的系统性坏死性血管炎和新月体肾小球肾炎。第一个具体目标侧重于ANCA诱导的中性粒细胞/单核细胞活化的机制。拟定的体外和体内研究是互补的，将平行进行。体外研究将通过处理来自健康个体的细胞来检查抗体的F（ab '）2部分相对于Fc部分的相对贡献。我们将设计抗PR 3和MPO F（ab '）2样分子，有和没有Fc区，以确定这一点。体内研究将研究来自患有活动性疾病、处于缓解期和复发期的患者的循环细胞的变化，即，检查ANCA抗原的表面表达，并检查与炎症相关的基因转录的变化。第二个特定目的将测试ANCA及其抗原PR 3和MPO对内皮的影响。这些研究将与具体目标1同时进行。我们已经知道PR 3和MPO可以转位到内皮细胞中，并且PR 3可以诱导内皮细胞凋亡，而MPO不诱导凋亡。我们将确定细胞进入的机制和对导致细胞凋亡的细胞信号通路的影响。通过缺失分析，我们将确定负责促凋亡活性的PR 3结构域。

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.