Neuroblastoma Cell Line and Lymphocytes Talk for Cladribine Influenced Apoptosis and Inflammation Pathways in Multiple Sclerosis (MS): An “In Vitro” Study (P2.2-095)

Abstract Objective: To study neurons response to therapy and to autoreactive cells by evaluating apoptotic and inflammation pathway in Healthy Donors (HDs) and multiple sclerosis (MS) patients (pts)’ lymphocytes and neuroblastoma cell line (SHSY5Y) co-cultures with and without CdA stimulation “in vitro”. Background: MS is characterized by autoreactive lymphocytes and inflammation in the Central Nervous System (CNS). In this context, many therapies act to defeat the mechanism but few of them are able to cross the Blood Brain Barrier (BBB). Cladribine (CdA) is a purine analogue molecule able to pass BBB, but no data available about its effects in CNS and on neurons. Design/Methods: 20 HDs (43,6±20,1; 10F) and 20 naive MS pts (45,5±21,4; 11F) age and sex matched were enrolled. Co-cultures were assessed and stimulated with 20 nM of CdA for many time points (4 and 24 hours); co-cultures control (no CdA) were considered too. Flow cytometric analysis was used to assess apoptosis on both cellular types. Cellular lysates were analyzed for pro and anti-apoptotic molecules (Bax; Smac/DIABLO; Bcl-2; Cyt-c) in Western Blot. Co-culture supernatants (with/without CdA) were examinated for pro and anti-inflammatory cytokines (OPN; IL-6; TNF-α; IFN-γ; IL-4; IL-23) with ELISA test. Results: In vitro experiments showed an increase of MS lymphocytes apoptosis during CdA treatment, if compared to basal cultures (p≤0,0025) and HDs (p≤0,001). Expression of anti-apoptotic proteins was higher in MS compared to HDs (p≤0,0001) and reduced during treatment (p≤0,0001). The same was showed in HDs cells. Anti-inflammatory proteins were increased during CdA treatment in MS lymphocytes co-cultures supernatants if compared to basal (p≤0,001) while pro-inflammatory molecules were reduced (p≤0,0005). Neurons (alone/co-cultures), were not affected by therapy for apoptotic pattern, since pro-apoptotic proteins were not involved. Conclusions: These data suggest that CdA reduces the viability of peripheral and central lymphocytes by apoptosis. No apoptotic signal in neurons was detected. Disclosure: Dr. Ruggieri has nothing to disclose. Dr. Gargano has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Teva Pharmaceuticals. Dr. Mastrapasqua has nothing to disclose. Dr. Palazzo has nothing to disclose. Dr. Frigeri has nothing to disclose. Dr. Visconti has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Merck Serono. Dr. Paolicelli has nothing to disclose. Dr. Trojano has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Biogen, Novartis, Roche, Merck and Genzyme; has received speaker honoraria from Biogen Idec, Sanofi-Aventis, Merck, Teva, Genzyme and Novartis. Dr. Trojano has received research support from Biogen Idec, Merck Serono and Novartis.