Contribution of apoptosis onto pathogenesis of neuro-Behcet's disease

根据本实验室及其他研究人员的最新发现，我们认为中性粒细胞功能亢进参与了白塞病的发病机制和病理生理过程。神经-白塞氏病是神经-白塞氏病的一个亚类，以典型的粘膜-皮肤-生殖系统疾病为特征，累及中枢神经系统，预后差。在我对这项资助的提议中，我想知道中性粒细胞中的这种异常是由中性粒细胞本身的凋亡还是由免疫活性细胞（包括抗原呈递巨噬细胞或免疫调节T淋巴细胞）的凋亡引起的。为了证明细胞凋亡，建立了通过琼脂糖凝胶电泳上的DNA条带检测凋亡片段，并通过最初由Nicoletti建立的流式细胞术，等人，我还利用流式细胞仪和它们的单克隆抗体，在这些细胞上和细胞中显示凋亡相关分子，Fas抗原（CD 95），Fas配体和bcl-2，以及来自淋巴细胞mRNA的RT-PCR。我很难在细胞制备后很快看到中性粒细胞的生理性凋亡。我未能证明任何差异的中性粒细胞凋亡的患者和健康对照组之间的任何手段，我已经选择。接着，我将实验细胞从中性粒细胞转向T淋巴细胞，患者组淋巴细胞的DNA带型与对照组相似，但Fas和Fas配体的表达在患者组中显著增加。基于这些发现，我开始用神经白塞病患者的细胞进行实验。我发现很难招募患者，因为这种疾病的发病率很低，而且很难在生理性凋亡之前收集新鲜细胞。仅1例可检测到细胞凋亡，与其他无神经系统受累的患者无差异。我需要进一步的实验来建立所描述的假设。

Based on recent findings from our laboratory as well as from others, we believe that hyperfunction of neutrophils involves in pathogenesis and pathophysiology of Behcet's disease. Neuro-Behcet's disease is a subset of the disease, characterized by typical mucocutaneogenital disorders, involvement in central nervous system, and poor prognosis. In my proposal to this grant was to ask whether this abnormality in neutrophils is brought by apoptosis of neutrophils themselves or of immunocompetent cells including antigen presenting macrophges or immunoregulating Tlymphcytes.To demonstrate apoptosis, lestablished to detect apopttic fragments by DNA banding on agarose gel electrophoresis, and by flow cytometry originally established by Nicoletti, et al. I also utilised flow cytometer to demonstrate apoptosis related molecules, Fas antigen (CD 95), Fas ligand, and bcl-2, utilizing flow cytemeter and their monoclonal antibodies, on and in those cells, and RT-PCR from mRNA from lymphocytes.I had very hard time to see phisiological apoptosis in neutrophils quite quickly after cell preparation. I failed to demonstrate any difference of apoptosis of neutrophils between patients and healthy controls by any means which I had choose. Next I turned cells for experiments from neutrophils to T lymphocytes.DNA banding pattern of lymphocytes from patients were similar to those from controls, however the expression of Fas and Fas ligand was increased significantly in patients group.Based on those findings, I started to do experiments with cells from patients with neuro-Behcet's disease. I found it very difficult to recruit the patients because of very low incidence of the disease, and to collect fresh cells before physiological apoptosis. Only one case could be determined the apoptosis, and found no difference from other patients without neurological involvements. I need further experiment to establish hypothesis described.