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Effects of Systemic Immunosuppression on Islet Engraftment and Function Into a Subcutaneous Biocompatible Device
Effects of Systemic Immunosuppression on Islet Engraftment and Function Into a Subcutaneous Biocompatible Device S. Marzorati, N. Bocca, R.D. Molano, A.R. Hogan, M. Doni, L. Cobianchi, L. Inverardi, C. Ricordi, and A. Pileggi ABSTRACT The aim of this study was to explore the effect of sirolimus (Sir) and tacrolimus (Tac) on islets implanted into a subcutaneous (SC), prevascularized device in syngeneic rats. Animals received a 40-day treatment with Tac and Sir (alone or in combination) starting either on day 0 or 40 days after islet transplantation. Controls received no treatment. A 40-day washout period was performed after immunosuppression (IS). Glycemia and intravenous glucose tolerance tests (IVGTT) were assessed at follow-up. In the control group, 75% of recipients achieved stable normoglycemia after islet transplantation, while none reversed diabetes with any IS regimen started on day 0. Graft dysfunction was irreversible after IS withdrawal. Glucose clearance (IVGTT) was significantly impaired among Tac-treated compared with control groups (P ⬍ .05 with IS; P ⬍ .01 after washout). Among animals with established grafts, islet dysfunction which occurred under IS treatment persisted after washout in animals treated with Tac and Sir plus Tac. When compared with controls, glucose clearance was significantly impaired in the Tac and Tac plus Sir groups before and after IS (P ⬍ .01, Tac; P ⬍ 0.01, Tac plus Sir). Sir and Tac showed profound deleterious effects on islet cell engraftment and function, which may hinder the success of implantation into biohybrid devices. Nondiabetogenic IS protocols must be developed for clinical application of islet transplantation into biohybrid devices.
R
ECENT DATA SUGGEST that immunosuppressive (IS) agents may adversely affect the engraftment and function of transplanted islets.1 Tacrolimus (Tac) is known for its diabetogenic toxicity leading to progressive islet cell dysfunction. Other data have shown that sirolimus (Sir) may adversely affect islet engraftment and compromise ß-cell function.2 The aim of this study was to explore the effect of Sir and Tac IS on islets implanted into a subcutaneous (SC), prevascularized device.3
additional experiment used the same IS protocol in animals with established syngeneic islet graft function (⬎40 days) prior to initiation of IS therapy. For both groups, we performed a 40-day washout period without IS drugs. Nonfasting glycemia (⬍200 mg/dL) and intravenous glucose tolerance tests (IVGTT) were performed to monitor graft function over time, as previously described.3
RESULTS
MATERIALS AND METHODS
In the control group, 75% of recipients maintained longterm normoglycemia after transplantation. When started at
Lewis rats (Harlan Laboratories) were used as both islet donors and recipients in compliance with the Institutional Animal Care and Use Commitee A biohybrid device was implanted SC into Lewis rats 40 days prior to islet transplantation to allow prevascularization of the site, as previously described.3 Islet isolation was performed using Liberase (Roche) digestion followed by Euroficoll density purification (Mediatech-Cellgro), as previously reported.3 To test the effects of the drugs on islet engraftment, animals received a 40-day treatment with Tac (daily SC at 1.0 mg/kg) or Sir (oral 3.0 mg/kg on days 0, 1, 2, and every other day thereafter) or the drug combination. Control animals received no treatment. An
From the Diabetes Research Institute (S.M., N.B., R.D.M., A.R.H., M.D., L.C., L.I., C.R., A.P.), University of Miami Miller School of Medicine, Miami, Florida, USA, and Department of Surgery (M.D., L.C.), Fondazione IRCCS “San Matteo” Hospital, University of Pavia, Pavia, Italy. This work was supported by Converge Biotechnologies, Inc, and the Diabetes Research Institute Foundation. Address reprint requests to Antonello Pileggi, MD, PhD, 1450 NW 10th Ave, Miami, FL 33136, USA. E-mail: apileggi@med. miami.edu
0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2008.09.057 352
© 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 352–353 (2009)
EFFECTS OF SIR/TAC ON ISLET CELL ENGRAFTMENT
the time of islet transplantation, animals under any IS regimen did not reverse their diabetes over a 40-day treatment. Graft dysfunction was not reversible after IS withdrawal. In the Sir group, only 33% of animals had transient graft dysfunction during IS treatment that resolved during the washout period. We observed a persistent significant difference in glucose clearance during IVGTT between control and Tac groups (P ⬍ .05 with IS; P ⬍ .01 after washout). In animals with established graft function, graft dysfunction (elevation of nonfasting glycemic values) was observed during the IS treatment; dysfunction persisted after washout in 66% and 100% of animals treated with Tac or Sir plusTac, respectively. In the Sir group, only 33% of animals had transient graft dysfunction during IS that resolved during the washout period. When compared with controls, significant differences in glucose clearance during IVGTT were observed in the Tac and Tacplus Sir groups before and after IS (P ⬍ .01, Tac; P ⬍ 0.01, TacplusSir).
353
DISCUSSION
Our data indicated that Sir and Tac have profound deleterious effects on islet cell engraftment and function, which may hinder the success of implantation into biohybrid devices. Nondiabetogenic IS protocols must be developed for clinical application of islet transplantation into biohybrid devices.4 REFERENCES 1. Pileggi A, Cobianchi L, Inverardi L, et al: Overcoming the challenges now limiting islet transplantation: a sequential, integrated approach. Ann NY Acad Sci 1079:383, 2006 2. Zahr E, Molano RD, Pileggi A, et al: Rapamycin impairs in vivo proliferation of islet beta-cells. Transplantation 84:1576, 2007 3. Pileggi A, Molano RD, Ricordi C, et al: Reversal of diabetes by pancreatic islet transplantation into a subcutaneous, neovascularized device. Transplantation 81:1318, 2006 4. Bocca N, Pileggi A, Molano RD, et al: Soft corticosteroids for local immunosuppression: exploring the possibility for the use of loteprednol etabonate for islet transplantation. Pharmazie 63:226, 2008
R
ECENT DATA SUGGEST that immunosuppressive (IS) agents may adversely affect the engraftment and function of transplanted islets.1 Tacrolimus (Tac) is known for its diabetogenic toxicity leading to progressive islet cell dysfunction. Other data have shown that sirolimus (Sir) may adversely affect islet engraftment and compromise ß-cell function.2 The aim of this study was to explore the effect of Sir and Tac IS on islets implanted into a subcutaneous (SC), prevascularized device.3
additional experiment used the same IS protocol in animals with established syngeneic islet graft function (⬎40 days) prior to initiation of IS therapy. For both groups, we performed a 40-day washout period without IS drugs. Nonfasting glycemia (⬍200 mg/dL) and intravenous glucose tolerance tests (IVGTT) were performed to monitor graft function over time, as previously described.3
RESULTS
MATERIALS AND METHODS
In the control group, 75% of recipients maintained longterm normoglycemia after transplantation. When started at
Lewis rats (Harlan Laboratories) were used as both islet donors and recipients in compliance with the Institutional Animal Care and Use Commitee A biohybrid device was implanted SC into Lewis rats 40 days prior to islet transplantation to allow prevascularization of the site, as previously described.3 Islet isolation was performed using Liberase (Roche) digestion followed by Euroficoll density purification (Mediatech-Cellgro), as previously reported.3 To test the effects of the drugs on islet engraftment, animals received a 40-day treatment with Tac (daily SC at 1.0 mg/kg) or Sir (oral 3.0 mg/kg on days 0, 1, 2, and every other day thereafter) or the drug combination. Control animals received no treatment. An
From the Diabetes Research Institute (S.M., N.B., R.D.M., A.R.H., M.D., L.C., L.I., C.R., A.P.), University of Miami Miller School of Medicine, Miami, Florida, USA, and Department of Surgery (M.D., L.C.), Fondazione IRCCS “San Matteo” Hospital, University of Pavia, Pavia, Italy. This work was supported by Converge Biotechnologies, Inc, and the Diabetes Research Institute Foundation. Address reprint requests to Antonello Pileggi, MD, PhD, 1450 NW 10th Ave, Miami, FL 33136, USA. E-mail: apileggi@med. miami.edu
0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2008.09.057 352
© 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 352–353 (2009)
EFFECTS OF SIR/TAC ON ISLET CELL ENGRAFTMENT
the time of islet transplantation, animals under any IS regimen did not reverse their diabetes over a 40-day treatment. Graft dysfunction was not reversible after IS withdrawal. In the Sir group, only 33% of animals had transient graft dysfunction during IS treatment that resolved during the washout period. We observed a persistent significant difference in glucose clearance during IVGTT between control and Tac groups (P ⬍ .05 with IS; P ⬍ .01 after washout). In animals with established graft function, graft dysfunction (elevation of nonfasting glycemic values) was observed during the IS treatment; dysfunction persisted after washout in 66% and 100% of animals treated with Tac or Sir plusTac, respectively. In the Sir group, only 33% of animals had transient graft dysfunction during IS that resolved during the washout period. When compared with controls, significant differences in glucose clearance during IVGTT were observed in the Tac and Tacplus Sir groups before and after IS (P ⬍ .01, Tac; P ⬍ 0.01, TacplusSir).
353
DISCUSSION
Our data indicated that Sir and Tac have profound deleterious effects on islet cell engraftment and function, which may hinder the success of implantation into biohybrid devices. Nondiabetogenic IS protocols must be developed for clinical application of islet transplantation into biohybrid devices.4 REFERENCES 1. Pileggi A, Cobianchi L, Inverardi L, et al: Overcoming the challenges now limiting islet transplantation: a sequential, integrated approach. Ann NY Acad Sci 1079:383, 2006 2. Zahr E, Molano RD, Pileggi A, et al: Rapamycin impairs in vivo proliferation of islet beta-cells. Transplantation 84:1576, 2007 3. Pileggi A, Molano RD, Ricordi C, et al: Reversal of diabetes by pancreatic islet transplantation into a subcutaneous, neovascularized device. Transplantation 81:1318, 2006 4. Bocca N, Pileggi A, Molano RD, et al: Soft corticosteroids for local immunosuppression: exploring the possibility for the use of loteprednol etabonate for islet transplantation. Pharmazie 63:226, 2008