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

根据我们实验室以及其他实验室的最新发现，我们认为中性粒细胞功能亢进与白塞病的发病机制和病理生理有关。神经白塞病是白塞病的一个亚型，以典型的粘液生殖障碍为特征，累及中枢神经系统，预后差。我对这笔赠款的建议是询问中性粒细胞的这种异常是由中性粒细胞本身的凋亡引起的，还是由包括抗原呈递巨噬细胞或免疫调节T淋巴细胞在内的免疫活性细胞的凋亡引起的。为了证明细胞凋亡，建立了通过琼脂糖凝胶电泳法上的DNA条带和最初由Nicoletti等人建立的流式细胞术来检测凋亡片段。用流式细胞仪检测细胞表面和细胞内的Fas抗原(CD95)、Fas配体、bc1-2等凋亡相关分子，并用RT-PCR方法检测淋巴细胞的mRNA表达。我没有通过我选择的任何方法来证明患者和健康对照组中性粒细胞凋亡的任何差异。接下来，我将细胞从中性粒细胞转变为T淋巴细胞。患者淋巴细胞的DNA带型与对照组相似，但患者组Fas和Fas配体的表达明显增加。基于这些发现，我开始用神经白塞病患者的细胞进行实验。我发现招募患者非常困难，因为这种疾病的发病率很低，而且在生理性细胞凋亡之前收集新鲜细胞。仅1例可检测到细胞凋亡，与其他无神经受累的患者无明显差异。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.