P-183

Establishment of a model of clinical islet transplantation in immunocompetent rats with intra-portal delivery of human islets and immunosuppression with sirolimus or tacrolimus

H.L. Marshall¹, S.C. Campbell¹, S. Van Cruchten², R. Arya¹, S.A. White³, C. Huggins¹, R. Stewart¹, A. Aldibbiat¹, L. Ferguson¹, T. Tree⁴, T. Allsopp⁵, A. Ting⁶, J.A.M. Shaw^1
1 Newcastle University, Newcastle upon Tyne, U.K.; ² Regenesys, Leuven, Belgium; ³ Institute of Transplantation, Freeman Hospital, Newcastle upon Tyne, U.K.; ⁴ King's College London, London, U.K.; ⁵ Pfizer Regenerative Medicine, Cambridge, U.K.; ⁶ Athersys, Inc., Cleveland, USA

Objectives: Intra-portal islet allografts are often associated with sub-optimal initial engraftment and relatively rapid attrition in graft function in comparison to vascularised pancreas transplantation. Elucidation of underlying mechanisms has been hampered by limited clinical histological data and absence of a suitable biomarker for early graft loss/ appropriate modalities for in vivo graft imaging. Our aim was to validate a rodent model of intra-portal islet transplantation using human islets to explore underlying mechanisms of early graft failure.

Methods: Diabetes was induced in Wistar rats weighing 360±33g by intraperitoneal streptozotocin injection (60mg/kg). Hepatic intra-portal vein infusion of deceased donor human islets (2000-3000IEQ/per recipient) was undertaken following laparotomy under general anaesthesia. Immunosuppression comprised either sirolimus (2mg/kg) every 48hrs or alemtuzumab induction (0.3mg/kg) with daily tacrolimus (0.1-0.2mg/kg). Clinical score and weight was recorded daily with monitoring of full blood count and trough tacrolimus levels. Weekly intra-peritoneal glucose tolerance tests (IPGTT) were undertaken (2g/kg dextrose). Blood glucose was measured by validated rat glucometer with plasma rat and human insulin (fasting/ 2 hours post-IPGTT) determined by sensitive differential ELISA.

Results: Irreversible diabetes was confirmed pre-transplantation (fasting glucose 23±3.4 mmol/l; 2 hour IPGTT 32.2±3.8 (n=12; mean±SD)). Reproducible intra-portal transplantation was achieved without haemorrhage/ other surgical complications. Immunosuppression was tolerated with few adverse effects. Glucose lowering was achieved in selected recipients (lowest fasting glucose 4.8 mmol/l; 2 hour IPGTT 18.8 mmol/l) with significant circulating human insulin levels. Absence of pancreas beta-cell regeneration was confirmed at post mortem. Increased serum cytokine levels were detected post-streptozotocin dosing with selected animals showing a secondary peak following transplantation.

Conclusions: A model encompassing all aspects of clinical islet transplantation has been established. Analysis of serum cytokines in parallel with pathological examination of graft constitution, inflammation and vascularisation will provide new insights into mechanisms underlying poor engraftment/ functional attrition and clinically important circulating biomarkers.

P-183

Establishment of a model of clinical islet transplantation in immunocompetent rats with intra-portal delivery of human islets and immunosuppression with sirolimus or tacrolimus

H.L. Marshall¹, S.C. Campbell¹, S. Van Cruchten², R. Arya¹, S.A. White³, C. Huggins¹, R. Stewart¹, A. Aldibbiat¹, L. Ferguson¹, T. Tree⁴, T. Allsopp⁵, A. Ting⁶, J.A.M. Shaw^1
1 Newcastle University, Newcastle upon Tyne, U.K.; ² Regenesys, Leuven, Belgium; ³ Institute of Transplantation, Freeman Hospital, Newcastle upon Tyne, U.K.; ⁴ King's College London, London, U.K.; ⁵ Pfizer Regenerative Medicine, Cambridge, U.K.; ⁶ Athersys, Inc., Cleveland, USA

Objectives: Intra-portal islet allografts are often associated with sub-optimal initial engraftment and relatively rapid attrition in graft function in comparison to vascularised pancreas transplantation. Elucidation of underlying mechanisms has been hampered by limited clinical histological data and absence of a suitable biomarker for early graft loss/ appropriate modalities for in vivo graft imaging. Our aim was to validate a rodent model of intra-portal islet transplantation using human islets to explore underlying mechanisms of early graft failure.

Methods: Diabetes was induced in Wistar rats weighing 360±33g by intraperitoneal streptozotocin injection (60mg/kg). Hepatic intra-portal vein infusion of deceased donor human islets (2000-3000IEQ/per recipient) was undertaken following laparotomy under general anaesthesia. Immunosuppression comprised either sirolimus (2mg/kg) every 48hrs or alemtuzumab induction (0.3mg/kg) with daily tacrolimus (0.1-0.2mg/kg). Clinical score and weight was recorded daily with monitoring of full blood count and trough tacrolimus levels. Weekly intra-peritoneal glucose tolerance tests (IPGTT) were undertaken (2g/kg dextrose). Blood glucose was measured by validated rat glucometer with plasma rat and human insulin (fasting/ 2 hours post-IPGTT) determined by sensitive differential ELISA.

Results: Irreversible diabetes was confirmed pre-transplantation (fasting glucose 23±3.4 mmol/l; 2 hour IPGTT 32.2±3.8 (n=12; mean±SD)). Reproducible intra-portal transplantation was achieved without haemorrhage/ other surgical complications. Immunosuppression was tolerated with few adverse effects. Glucose lowering was achieved in selected recipients (lowest fasting glucose 4.8 mmol/l; 2 hour IPGTT 18.8 mmol/l) with significant circulating human insulin levels. Absence of pancreas beta-cell regeneration was confirmed at post mortem. Increased serum cytokine levels were detected post-streptozotocin dosing with selected animals showing a secondary peak following transplantation.

Conclusions: A model encompassing all aspects of clinical islet transplantation has been established. Analysis of serum cytokines in parallel with pathological examination of graft constitution, inflammation and vascularisation will provide new insights into mechanisms underlying poor engraftment/ functional attrition and clinically important circulating biomarkers.