CD95/CD95L DISEQUILIBRIUM IN PERIPHERAL BLOOD MONONUCLEAR CELLS IN MULTIPLE SCLEROSIS ACUTE PHASES.

Introduction. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of central nervous system (CNS). ln active disease a transmigration of autoreactive T cells to myelin antigens recruited from the peripheral blood to CNS occurs and contributes to the perpetuation of the inflammation. Our previous study (1) showed a peculiar impaired behaviour of apoptosis receptor complex CD95/CD95L on membrane of peripheral blood mononuclear cells (PBMC) of MS patients: CD95L decreased dramatically while CD95 increased in active disease, instead both receptor and ligand increased in stable disease. The aim of this study was to evaluate mFlNA levels and protein expression of CD95/CD95L in PBMC of MS patients to find an eventual correlation with our previous data. Methods. 40 MS patients, 25 stable and 15 active, 14 male and 26 female, aged 15-55 years, were enrolled. 10 aged and sex matched healthy controls were used. PBMC were isolated and resuspended in Trizol reagent for extraction of total RNA. Quality of the extracted RNA was evaluated by formamide/agarose electrophoresis and quantity was determined by spectrophotometer at OD250. Only samples with a ratio 31.7 were used for the successive semiquantitative analysis by reverse transcriptase (FtT)—PCR. Proteins were extracted by RIPA buffer, dissolved in 1% SDS and analyzed by spectrophotometer at OD75O using a colorimetric assay. The cell lysates (40 »g of protein) were resolved by SDS PAGE and blotted on PVDF membrane. The samples were probing with appropriate primary Ab and beta—actin. The secondary Abs were HRP-conjugated. Signals were detected using ECL system. Results and Discussion. Our results showed high mFlNA levels for both CD95 and CD95L in both active and stable patients vs controls. CD95 protein expression confirmed data obtained by RT-PCR in all samples, while CD95L protein dramatically decreased only in active MS patients. These data confirm the impairment on membrane expression previously observed and let us hypothesize that post—transcriptional modifications occur in active phase of disease.