Contributions
Abstract: EP1608
Type: Poster Sessions
Abstract Category: Therapy - Immunomodulation/Immunosuppression
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 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.
Aim: We evaluated apoptotic and inflammation pathway in Healthy Donors (HDs) and MS patients lymphocytes and neuroblastoma cell line (SHSY5Y) co-cultures with and without CdA stimulation “in vitro”, in order to study neurons response to therapy and to autoreactive cells.
Methods: 20 HDs (43,6±20,1; 10F) and 20 naive MS pts (45,5±21,4; 11F) age and sex matched were enrolled in our study. 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, at first, 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 (stimulated or not with CdA) were examinated for pro and anti-inflammatory cytokines (OPN; IL-6; TNF-; 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 related to HDs (p≤0,001). Anti-apoptotic proteins showed higher expression in MS if compared to HDs (p≤0,0001) and it was 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 or in co-cultures, were not affected by therapy for apoptotic pattern, since pro-apoptotic proteins were not involved.
Conclusions: These data suggest that CdA likely reduces the viability of peripheral and central lymphocytes by apoptosis. No apoptotic signal in neurons was detected.
Disclosure: Prof. Maria Trojano has served on scientific Advisory Boards for Biogen, Novartis, Roche and Genzyme; has received speaker honoraria from Biogen Idec, Sanofi-Aventis, Merck Serono, Teva, Genzyme and Novartis; and has received research grants for her Institution from Biogen Idec, Merck Serono and Novartis.
Dr Paolicelli has received honoraria for consultancy and/or speaking from Biogen Idec, Merck-Serono, Sanofi-Aventis, TEVA, Bayer-Schering, Novartis and Genzyme.
MR, MM, CDG, CP, AF, AV have nothing to disclose.
Abstract: EP1608
Type: Poster Sessions
Abstract Category: Therapy - Immunomodulation/Immunosuppression
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 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.
Aim: We evaluated apoptotic and inflammation pathway in Healthy Donors (HDs) and MS patients lymphocytes and neuroblastoma cell line (SHSY5Y) co-cultures with and without CdA stimulation “in vitro”, in order to study neurons response to therapy and to autoreactive cells.
Methods: 20 HDs (43,6±20,1; 10F) and 20 naive MS pts (45,5±21,4; 11F) age and sex matched were enrolled in our study. 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, at first, 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 (stimulated or not with CdA) were examinated for pro and anti-inflammatory cytokines (OPN; IL-6; TNF-; 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 related to HDs (p≤0,001). Anti-apoptotic proteins showed higher expression in MS if compared to HDs (p≤0,0001) and it was 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 or in co-cultures, were not affected by therapy for apoptotic pattern, since pro-apoptotic proteins were not involved.
Conclusions: These data suggest that CdA likely reduces the viability of peripheral and central lymphocytes by apoptosis. No apoptotic signal in neurons was detected.
Disclosure: Prof. Maria Trojano has served on scientific Advisory Boards for Biogen, Novartis, Roche and Genzyme; has received speaker honoraria from Biogen Idec, Sanofi-Aventis, Merck Serono, Teva, Genzyme and Novartis; and has received research grants for her Institution from Biogen Idec, Merck Serono and Novartis.
Dr Paolicelli has received honoraria for consultancy and/or speaking from Biogen Idec, Merck-Serono, Sanofi-Aventis, TEVA, Bayer-Schering, Novartis and Genzyme.
MR, MM, CDG, CP, AF, AV have nothing to disclose.