ABSTRACT
Despite recent successes in islet transplantation, current immunosuppression protocols required to prevent graft rejection are not suitable for all patients. As a consequence, microencapsulation of islets in alginate has been proposed to protect islets from immune-mediated destruction. Success has been limited, however, due largely to problems with alginate biocompatibility and insufficient immunoprotection by the capsule. The aim of this study was to develop a purified, highly biocompatible, and highly stable alginate from commercially available alginate. We analyzed the chemical properties of the alginate before and after purification and compared in vivo survival and metabolic function of mouse islets encapsulated with either alginate in syngeneic recipients. Recipients of purified alginate capsules exhibited a 105-day graft survival rate of 90.5%, versus 69.2% for recipients of nonpurified alginate, with recipients of purified alginate capsules also showing improved nonfasting blood glucose levels and oral glucose challenges over recipients of nonpurified alginate. On recovery, islets encapsulated in purified alginate capsules demonstrated dramatically reduced capsular overgrowth, and an insulin secretory activity far superior to that of islets in nonpurified alginate capsules. We conclude from this study that alginate purification improves the survival and metabolic function of encapsulated islets. To our knowledge, this is the first paper using pre- and postmodification alginate to demonstrate the direct benefit of purification on transplantation success of islets in simple, open-pore capsules.
Subject(s)
Alginates/metabolism , Graft Survival , Islets of Langerhans Transplantation , Alginates/chemistry , Alginates/isolation & purification , Animals , Capsules , Glucose Tolerance Test , Glucuronic Acid/chemistry , Glucuronic Acid/isolation & purification , Glucuronic Acid/metabolism , Hexuronic Acids/chemistry , Hexuronic Acids/isolation & purification , Hexuronic Acids/metabolism , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred BALB C , Time FactorsABSTRACT
BACKGROUND: The immune mechanisms associated with the rejection of microencapsulated neonatal porcine islets (NPI) are not clearly understood. Therefore, in this study we characterized the immune cells and molecules that are involved in this process by examining the microencapsulated NPI xenografts at various time points post-transplantation in B6 mice. METHODS: Microencapsulated NPI were transplanted into streptozotocin-induced diabetic immune-competent B6 and immune-deficient B6 rag-/- mice and blood glucose levels were monitored twice a week. Encapsulated NPI were then recovered from B6 mice at various time points post-transplantation to characterize the islets and immune response using immunohistochemical and RT-PCR analyses. To determine which T-cell subpopulation is important for the rejection of encapsulated NPI, B6 rag-/- mice with established microencapsulated NPI xenografts were reconstituted with either CD4(+) or CD8(+) T cells and a return to the diabetic state was noted. For controls, adoptive transfer experiments involved reconstitution of B6 rag-/- mice with established microencapsulated NPI with non-fractionated lymph node cells or non-reconstituted mice. RESULTS: All B6 recipients of microencapsulated NPI remained diabetic throughout the study while B6 rag-/- recipients achieved normoglycemia and maintained normoglycemia for up to 100 days post-transplantation. Encapsulated NPI recovered from B6 mice at early time points (day 7 and day 14) post-transplantation were surrounded with very few layers of immune cells that increased with time post-transplantation. The extent of cellular overgrowth on the surface of encapsulated NPI has a significant correlation with islet cell death and the presence of CD4(+) T cells, B cells and macrophages. Mouse IgG antibody and complement as well as cytokines [gamma-interferon (IFN-gamma), interleukin10 (IL10)] and chemokines (monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha and beta) were detected within the microcapsules at several time points post-transplantation suggesting that these molecules can traverse the microcapsule. Mouse anti-porcine IgG antibodies in recipient sera were found to peak at 30 days post-transplantation indicating leakage of porcine xenoantigens. In contrast, microencapsulated NPI recovered from B6 rag-/- mice had no cellular overgrowth on the surface. Complement and cytokines (IL 10 but not IFN-gamma) including chemokines were detected within the microcapsules at several days post-transplantation. We also found that B6 rag-/- mice reconstituted with non-fractionated lymph node cells or CD4(+) T cells but not CD8(+) T cells became diabetic demonstrating that CD4(+) T cells are the necessary T-cell subtype for microencapsulated NPI rejection. In contrast, non-reconstituted B6 rag-/- mice remained normoglycemic for the entire duration of the study. CONCLUSIONS: Our results demonstrate that CD4(+) T cells, B cells and macrophages are the immune cells recruited to and involved in the rejection of encapsulated NPI. Immune molecules secreted by these cells as well as complement can traverse the microcapsule membrane and are responsible for destroying the NPI cells. Treatment regimens which target these molecules may modify the rejection of encapsulated NPI and lead to prolonged islet xenograft survival.
