- Email: [email protected]
Development of a unique in vivo murine model to study islet engraftment and function
S8
Abstracts
Sunday, October 3, 2004 2:00 PM – 3:30 PM Abstract Session #2: Islet Cell Transplant and Regulation of Immune Responses 1.1 8-OR
SIGNIFICANCE OF HLA IN HUMAN ISLET TRANSPLANTATION T. Mohanakumar,1,2 K. Narayanan,1 D. Phelan,4 B. Olack,1 N. Benshoff,1 S. Ramachandran,1 D. Brennan,3 J. Odorico,5 N. Desai,1 K. Polonsky3, 1 Surgery, Washington University; 2Pathology, Washington University; 3Internal Medicice, Washington University, St. Louis, MO, USA; 4HLA Laboratory, Barnes Hospital, St. Louis, MO, USA; 5Surgery, University of Wisconsin, Madison, WI, USA With improvements in isolation and immunosuppression, cellular transplantation (Tx) is becoming a reality. A general belief is that HLA plays only a minimal role in islet Tx. Our goals were; 1) determine the impact of HLA presensitization and islet function, and 2) determine whether post Tx monitoring for antibodies (Abs) to HLA can indicate rejection. Results: 7 recipients JDRF (4) and NIH/ICR (3) were assessed. One patient (Pt) had 3 islet Txs, 3 had 2, and 3 had 1. HLA matching was not done. Cytotoxicity and ELISA identified 1 Pt (JDRF 4) reactive to a panel. FLOW revealed 2 other sensitized Pts (JDRF1 and ICR2). Cytotoxicity crossmatch failed to detect any reactivity to the donors whereas FLOW identified 3 (JDRF1, JDRF3 and ICR2). Two of these (JDRF1 and ICR2) had sensitization only by FLOW and lost islet function within weeks. The third (JDRF4) who was sensitized but negative crossmatch lost islet function. Analysis revealed the presence of a cross-reactive antigen (HLA-B7). ELISPOT for IFN- following donor stimulation confirmed cellular sensitization in every case where Ab was defined by FLOW. Donor specific Ab following Tx showed that 2 Pts Tx’d with a negative crossmatch who lost function developed HLA Abs. In summary, cellular Tx follows the same rules as solid organ, ie, pre-existing donor specific HLA Abs are harmful and rejection of the islets is often preceded by the development of HLA Abs. These results have led us to use a positive crossmatch as a contraindication for Tx and to monitor for donor specific HLA Abs to predict islet rejection.
2 9-OR
DEVELOPMENT OF A UNIQUE IN VIVO MURINE MODEL TO STUDY ISLET ENGRAFTMENT AND FUNCTION A. Bharat,1 N. Benshoff,1 S. Ramachandran,1 N. Desai,1 T. Mohanakumar1,2, 1Surgery, Washington Uuniversity; 2Pathology, Washington University, St. Louis, MO, USA Introduction: Islet transplantation under kidney capsule (KC) in diabetic SCID (d-SCID) mice is the standard for in vivo islet function studies. However, poor neovascularization at this site leads to inconsistent islet function. Besides, this procedure is technically demanding and associated with significant animal mortality. Matrigel basement membrane matrix (MM), rich in angiogenic factors, is known to enhance neovascularisation. The goal of this study was to test if MM would be optimal for subcutaneous(s/c) islet transplantation. Methodology: Streptozotocin (200mg/kg, single IP) was used to induce diabetes (non-fasting blood glucose [NFBG] ⬎ 300mg/dl) in SCID mice. 3000 human islet equivalents (HIE) were either injected s/c with MM or transplanted under KC. NFBG was monitored for 30 days following which grafts were retrieved. Similarly, 3000 porcine islet equivalents (PIE) were injected s/c with MM into d-SCID mice. Results: Seven of 9 mice in MM and 7 of 10 in KC group achieved normoglycemia (NFBG⬍150 mg/dl) with 3000 HIE. MM group revealed NFBG control much earlier (mean 3.4 days Vs 5.3, p⫽0.03). All 7 recipients of 3000 PIE with MM achieved rapid normoglycemia. Upon graft removal, the animals became hyperglycemic again. Histology showed presence of discrete islets with well-preserved architecture and numerous blood vessels in the MM. In contrast, islets under KC appeared clumped and encroaching the renal parenchyma with no discernible vessels developing into the graft. Conclusions: Islets transplanted s/c with MM can restore normoglycemia. This model is an attractive alternate to KC transplantation and provides a unique opportunity to study islet engraftment since MM can be easily reconstituted with cytokines and growth factors.
Abstracts
Sunday, October 3, 2004 2:00 PM – 3:30 PM Abstract Session #2: Islet Cell Transplant and Regulation of Immune Responses 1.1 8-OR
SIGNIFICANCE OF HLA IN HUMAN ISLET TRANSPLANTATION T. Mohanakumar,1,2 K. Narayanan,1 D. Phelan,4 B. Olack,1 N. Benshoff,1 S. Ramachandran,1 D. Brennan,3 J. Odorico,5 N. Desai,1 K. Polonsky3, 1 Surgery, Washington University; 2Pathology, Washington University; 3Internal Medicice, Washington University, St. Louis, MO, USA; 4HLA Laboratory, Barnes Hospital, St. Louis, MO, USA; 5Surgery, University of Wisconsin, Madison, WI, USA With improvements in isolation and immunosuppression, cellular transplantation (Tx) is becoming a reality. A general belief is that HLA plays only a minimal role in islet Tx. Our goals were; 1) determine the impact of HLA presensitization and islet function, and 2) determine whether post Tx monitoring for antibodies (Abs) to HLA can indicate rejection. Results: 7 recipients JDRF (4) and NIH/ICR (3) were assessed. One patient (Pt) had 3 islet Txs, 3 had 2, and 3 had 1. HLA matching was not done. Cytotoxicity and ELISA identified 1 Pt (JDRF 4) reactive to a panel. FLOW revealed 2 other sensitized Pts (JDRF1 and ICR2). Cytotoxicity crossmatch failed to detect any reactivity to the donors whereas FLOW identified 3 (JDRF1, JDRF3 and ICR2). Two of these (JDRF1 and ICR2) had sensitization only by FLOW and lost islet function within weeks. The third (JDRF4) who was sensitized but negative crossmatch lost islet function. Analysis revealed the presence of a cross-reactive antigen (HLA-B7). ELISPOT for IFN- following donor stimulation confirmed cellular sensitization in every case where Ab was defined by FLOW. Donor specific Ab following Tx showed that 2 Pts Tx’d with a negative crossmatch who lost function developed HLA Abs. In summary, cellular Tx follows the same rules as solid organ, ie, pre-existing donor specific HLA Abs are harmful and rejection of the islets is often preceded by the development of HLA Abs. These results have led us to use a positive crossmatch as a contraindication for Tx and to monitor for donor specific HLA Abs to predict islet rejection.
2 9-OR
DEVELOPMENT OF A UNIQUE IN VIVO MURINE MODEL TO STUDY ISLET ENGRAFTMENT AND FUNCTION A. Bharat,1 N. Benshoff,1 S. Ramachandran,1 N. Desai,1 T. Mohanakumar1,2, 1Surgery, Washington Uuniversity; 2Pathology, Washington University, St. Louis, MO, USA Introduction: Islet transplantation under kidney capsule (KC) in diabetic SCID (d-SCID) mice is the standard for in vivo islet function studies. However, poor neovascularization at this site leads to inconsistent islet function. Besides, this procedure is technically demanding and associated with significant animal mortality. Matrigel basement membrane matrix (MM), rich in angiogenic factors, is known to enhance neovascularisation. The goal of this study was to test if MM would be optimal for subcutaneous(s/c) islet transplantation. Methodology: Streptozotocin (200mg/kg, single IP) was used to induce diabetes (non-fasting blood glucose [NFBG] ⬎ 300mg/dl) in SCID mice. 3000 human islet equivalents (HIE) were either injected s/c with MM or transplanted under KC. NFBG was monitored for 30 days following which grafts were retrieved. Similarly, 3000 porcine islet equivalents (PIE) were injected s/c with MM into d-SCID mice. Results: Seven of 9 mice in MM and 7 of 10 in KC group achieved normoglycemia (NFBG⬍150 mg/dl) with 3000 HIE. MM group revealed NFBG control much earlier (mean 3.4 days Vs 5.3, p⫽0.03). All 7 recipients of 3000 PIE with MM achieved rapid normoglycemia. Upon graft removal, the animals became hyperglycemic again. Histology showed presence of discrete islets with well-preserved architecture and numerous blood vessels in the MM. In contrast, islets under KC appeared clumped and encroaching the renal parenchyma with no discernible vessels developing into the graft. Conclusions: Islets transplanted s/c with MM can restore normoglycemia. This model is an attractive alternate to KC transplantation and provides a unique opportunity to study islet engraftment since MM can be easily reconstituted with cytokines and growth factors.