P-235

IBMIR during islet transplantation: The role of the Toll-like Receptors signaling pathways

K. Vivot¹, A. Langlois¹, W. Bietiger¹, J.P. Gies¹, M. Pinget², S. Sigrist^1
1 Centre Européen d'étude du Diabète, Starsbourg, France; ² Université de Strasbourg , Strasbourg, France

Although islet transplantation is an attractive treatment for Type 1 diabetes, the not fully understood instant blood-mediated inflammatory reaction (IBMIR) leads to disappointing outcomes. This inflammatory process led to a massive destruction of transplanted islets immediately after transplantation. Islet isolation and time of culture could participate to the initial inflammatory response. The events before transplantation could elicit the release of potent activators of innate immune system by the Toll-Like Receptors (TLRs) downstream signaling pathways and result in IBMIR. This work aimed to evaluate in vitro the role of pre-transplantation proceedings in the mediation of inflammatory reaction by the study of the TLRs signaling pathways. Rat pancreatic islets were isolated and cultured during 24 hours or 48 hours. The viability was assessed by FDA/PI and the functionality of islets was evaluated using glucose stimulation test. After ARN extraction and retrotranscription, PCR arrays were performed. Extracts harvested immediately after isolation were defined as control samples. Eighty one genes, belonging to the TLRs signaling pathways, were screened and analyzed in comparison to control samples. Whatever the condition analyzed, islets were always living and functional. Concerning the PCRarrays analysis, significant up-regulation of TLR-2 was highlighted. After 24 hours of islet culture, TLR-2 was 7.7 fold (p<0,001) up-regulated compared to control but decreased to 4.5 fold after 48 hours. On the contrary, from 24 hours TLR-4 gene was significantly down-regulated to 2.2 fold (p<0.01) compared to control. The interleukin-6 gene expression was dramatically up-regulated to 95.3 fold compared to control. With the same way, the gene expression of cyclooxygenase-2 was up-regulated to reach 363.5 fold after 24 hours of islet culture. These preliminary data seem highlighted that TLRs signaling pathways would be implicated in early inflammatory events leading to islet destruction. Targeting the TLRs signaling could be relevant to improve islet transplantation.

P-235

IBMIR during islet transplantation: The role of the Toll-like Receptors signaling pathways

K. Vivot¹, A. Langlois¹, W. Bietiger¹, J.P. Gies¹, M. Pinget², S. Sigrist^1
1 Centre Européen d'étude du Diabète, Starsbourg, France; ² Université de Strasbourg , Strasbourg, France

Although islet transplantation is an attractive treatment for Type 1 diabetes, the not fully understood instant blood-mediated inflammatory reaction (IBMIR) leads to disappointing outcomes. This inflammatory process led to a massive destruction of transplanted islets immediately after transplantation. Islet isolation and time of culture could participate to the initial inflammatory response. The events before transplantation could elicit the release of potent activators of innate immune system by the Toll-Like Receptors (TLRs) downstream signaling pathways and result in IBMIR. This work aimed to evaluate in vitro the role of pre-transplantation proceedings in the mediation of inflammatory reaction by the study of the TLRs signaling pathways. Rat pancreatic islets were isolated and cultured during 24 hours or 48 hours. The viability was assessed by FDA/PI and the functionality of islets was evaluated using glucose stimulation test. After ARN extraction and retrotranscription, PCR arrays were performed. Extracts harvested immediately after isolation were defined as control samples. Eighty one genes, belonging to the TLRs signaling pathways, were screened and analyzed in comparison to control samples. Whatever the condition analyzed, islets were always living and functional. Concerning the PCRarrays analysis, significant up-regulation of TLR-2 was highlighted. After 24 hours of islet culture, TLR-2 was 7.7 fold (p<0,001) up-regulated compared to control but decreased to 4.5 fold after 48 hours. On the contrary, from 24 hours TLR-4 gene was significantly down-regulated to 2.2 fold (p<0.01) compared to control. The interleukin-6 gene expression was dramatically up-regulated to 95.3 fold compared to control. With the same way, the gene expression of cyclooxygenase-2 was up-regulated to reach 363.5 fold after 24 hours of islet culture. These preliminary data seem highlighted that TLRs signaling pathways would be implicated in early inflammatory events leading to islet destruction. Targeting the TLRs signaling could be relevant to improve islet transplantation.