From the DEPARTMENT OF CLINICAL SCIENCE, INTERVENTION AND TECHNOLOGY DIVISION OF TRANSPLANTATION SURGERY Karolinska Institutet, Stockholm, Sweden STRATEGIES TO IMPROVE MACROENCAPSULATED ISLET GRAFT SURVIVAL

Chronic immunosuppressive therapy may have severe side-effects. In cell transplantation, the graft can be encapsulated within a membrane chamber, providing a physical barrier against the immune system. The cell graft then becomes dependent on the diffusion of nutrients and oxygen from the surrounding microcirculation. A major drawback has been the formation of avascular fibrotic tissue around the chamber. The immunoprotective device studied (TheraCyteTM) has an outer membrane inducing neovascularization. However, major parts of the encapsulated graft are still lost soon after transplantation, probably because of relative hypoxia and malnutrition. The overall aim of this thesis was to assess various strategies to improve islet graft survival in the device, using rodent models. The purpose of the first paper was to improve the method for histological evaluation of the vascularization around the device. Vascular profiles within various distances from the membrane surface were counted at different times and then correlated with glucose kinetics. We found that the vascular profiles within 100 μm had the highest correlations with glucose kinetics and concluded that vessels within this distance are important for the exchange of small molecules between the circulation and the device’s lumen. Therefore, we recommend that 100 μm should be used in histological evaluations of the membrane vascularization. In the second paper we hypothesized that preimplantation of the device should improve encapsulated islet graft survival. Previous studies have indicated that it takes up to 3 months for recovery of the microcirculation after membrane implantation. Therefore, we implanted empty devices and transplanted islets 3 months later in these chambers. This approach significantly improved the cure rates of diabetic animals, and the islet dose required for cure was reduced by about 10 times. Morphometry evaluations confirmed increased graft survival in preimplanted devices. The third paper aimed at evaluating the effects of exendin-4 treatment on the metabolic outcome after islet transplantation. Exendin-4 inhibits islet apoptosis, stimulates islet differentiation and regeneration and has beneficial effects on peripheral tissues. We found that exendin-4 treatment significantly improved the metabolic outcome after free islet transplantation to the renal subcapsular site. The benefit lasted longer than the treatment, suggesting that exendin-4 had long-standing effects on the islet graft. This substance seems to be an interesting new approach to improve the survival also of encapsulated islet grafts. In the last paper we evaluated the risk of recipient sensitization using macroencapsulated islets. A heterotopic heart graft was transplanted one month after free or encapsulated islet transplantation. The time-to-rejection was significantly shorter in the free islet group, while it did not differ between encapsulated islet graft recipients and naive animals. We therefore conclude that the device protects against sensitization, at least during the first month after transplantation. Today, side-effects of the immunosuppressive therapy are one of the main limiting factors for the use of islet transplantation. If immunoprotection could be achieved by encapsulation of the islet graft, it should be possible to widen the indications. This thesis describes promising strategies to improve the survival of macroencapsulated islet grafts, which might contribute to make macroencapsulation a clinical reality. LIST OF PUBLICATIONS This thesis is based on the following papers which will be referred to by their Roman numerals: I. Improved histological evaluation of vascularity around an immunoisolation device by correlating number of vascular profiles to glucose exchange. Sörenby A, Rafael E, Tibell A, Wernerson A, Cell Transplantation 2004, Vol 13, 713-19. II. Preimplantation of an immunoprotective device can lower the curative dose of islets to that of free islet transplantation – studies in a rodent model. Sörenby A, Kumagai-Braesch M, Sharma A, Hultenby K, Wernerson A, Tibell A. Submitted. III. Exendin-4 treatment improves metabolic control after rat islet transplantation to athymic mice with streptozotocin-induced diabetes. Sharma A, Sörenby A, Wernerson A, Efendic S, Kumagai-Braesch M, Tibell A, Diabetologia, 2006, 49(6), 1247-53. IV. Macroencapsulation protects against sensitization after allogeneic islet transplantation in rats. Sörenby A, Wu G, Zhu S, Wernerson A, SumitranHolgersson S, Tibell A. Transplantation 2006, Vol 82(3), 393-7. Copyright Paper I: Cognizant Comm. Corp. Paper III: Springer-Verlag Paper IV: Lippincott Williams & Wilkins CONTENTS 1 Background. 1 1.1 -cell transplantation . 2 1.1.1 Pancreas transplantation . 2 1.1.2 Islet transplantation. 3 1.2 Side-effects of immunosuppressive drug therapy. 8 1.2. 1 Indications for islet transplantation. 9 1.3 Immunoprotection. 10 1.3.1 Common problems and improvement strategies . 11 1.3.2 Microencapsulation. 14 1.3. 3 Macroencapsulation. 16 1.3. 4 Previous studies of the bilayer membrane . 18 1.3. 5 Protection against sensitization of the recipient. 22 2 Specific Aims . 24 3 Materials and methods. 25 3.1 Materials, techniques and models . 25 3.1.1 The immunoprotective membrane. 25 3.1.2 The BoggsTM chamber . 25 3.1.3 The TheraCyte device. 26 3.1. 4 Rat islet isolation . 27 3.1. 5 Streptozotocin induction of diabetes. 28 3.1. 6 Transplantation models. 28 3.1. 7 Islet recipient management . 29 3.1. 8 Determination of anti-donor antibodies. 30 3.1. 9 Histological studies. 31 3.2 Designs of the individual studies . 33 3.2. 1 Paper I . 33 3.2. 2 Paper II. 35 3.2. 3 Paper III . 36 3.2. 4 Paper IV . 38 4 Results and discussion. 39 4.1 Paper I . 39 4.1.1 Discussion Paper I . 43 4.2 Paper II. 44 4.2. 1 Discussion Paper II . 49 4.3 paper III. 52 4.3.1 Discussion Paper III. 55 4.4 Paper IV . 57 4.4.1 Discussion Paper IV. 60 5 Conclusions . 65 6 Future prospects. 66 7 Acknowledgments . 68 8 References . 71 LIST OF ABBREVIATIONS APC Antigen-presenting cells AUC0-100 Area-under-the-curve, 0-100 minutes AUC0-40 Area-under-the-curve, 0-40 minutes AUC40-100 Area-under-the-curve, 40-100 minutes Bw Body weight CAT Catalase Cmax Maximum concentration CyA Cyclosporine DA Dark Agouti rats DSG 15-deoxyspergualine Encap. Encapsulated Ex-4 Exendin-4 GLP-1 Glucagon-like peptide-1 GPX Glutathione peroxidase H&E Hematoxylin and eosin HbA1c Glycosulated hemoglobin IBMIR Instant blood-mediated inflammatory reaction IEQ Islet equivalents IPGTT Intraperitoneal glucose tolerance test IPTR International Pancreas Transplant Registry IVGTT Intravenous glucose tolerance test MMF Mycophenolate mofetil NA Nicotinamide NO Nitric oxide ns Not significant PBS Phosphate-buffered saline PET Positron-emission tomography PI Preimplanted SD Sprague Dawley SOD Superoxide dismutase T1DM Type 1 Diabetes mellitus T2DM Type 2 Diabetes mellitus TTP Time-to-peak VEGF Vascular endothelial growth factor Vv’s Volume densities