|Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
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.