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P011. Resistance to Fas-apoptosis of peripheral T-cells from Crohn's disease patients depends on the regulation that catalase exerts on the apoptotic pathways

B. Beltrán1,2, I. Moret1, F. Rausell2, M. Iborra1, G. Bastida1,2, M. Aguas1, L. Tortosa1, P. Nos1,2

1Hospital Universitario la Fe/Instituto de Investigacion Sanitaria, Valencia, Spain; 2CIBERehd, Barcelona, Spain

Aims: As we have previously reported (JCC 2010, P016) resistance to apoptosis of T-lymphocytes from Crohn's Disease (CD) can be observed in primary cells, before they reach the intestinal mucosa. However, the apoptotic mechanisms implied in this resistance have not been yet clarified. We have aimed to study the survival and death pathways responsible of the apoptosis resistance observed.

Materials and Methods: Heparinised blood samples were taken from healthy subjects (n = 18) and from patients at onset of CD and yet to begin any specific medication (n = 18). Patients were diagnosed according to Leonard-Jones criteria and disease activity was scored based on the Harvey-Bradshaw index. Lymphocytes were isolated by Histopaque gradient centrifugation, followed by negative purification of non-activated T cells. Isolated cells were cultured for 5 days in the presence (to activate) or absence of bounded anti-CD3 and anti-CD28 diluted in X Vivo-15 medium. Afterwards, Fas antibody (Fas Ab) was added (1 μg/mL) and apoptosis was detected by flow-cytometry (Annexin-V) after overnight incubation. Western blot of NFkB subunits and Bax/Bcl-2 proteins were analysed from cell lysates by using specific antibodies to each protein. Also, analysis of NFkB were done by chemiluminiscence ELISA. Caspase activities (caspase 3, 8 and 9) were analysed by colorimetric assay kits. Catalase activity was also measured by a commercial kit.

Results: The addition of Fas Ab significantly increased the percentage of apoptotic control lymphocytes vs CD (P < 0.005). Analysis of NFkB subunits in these cells by western blot or chemiluminiscence ELISA gave rise to no differences between CD patients and controls (0.61 vs 0.43 densitometry units for p65). Also, similar results were obtained for Bax/Bcl-2 ratio (0.2742 vs 0.3931 densitometry units). Furthermore, none of caspases analysed showed different activities between CD patients and controls. Analysis of catalase showed a significant lower activity in patients than in controls (11.6 vs 3.3, P < 0.05).

Conclusions: Our results suggest that the classical apoptotic pathways (NFkB, Bax/Bcl-2 and caspase 3, 8 and 9) are not directly implicated in the apoptotic resistance observed, but the lower catalase activity is related to this process. Further investigation in this mechanism, already published in cell lines cultures, should be done to clarify the apoptotic resistance seen in CD lymphocytes.