Fetal pig islet-like cell clusters (ICCs) were transplanted into the thymus or omentum of STZ-induced diabetic pigs immunosuppressed with cyclosporine (CsA) and deoxyspergualin (DSG), as a potential treatment for type 1 diabetes. C-peptide levels in response to glucagon and arginine significantly increased over time using 1 litter of ICCs with highest levels obtained at 100 days post-transplantation. Increasing the number of ICCs to 2 litters was not advantageous. Histology of the graft showed all 4 pancreatic endocrine cells. Normoglycaemia was achieved for transient periods without insulin administration in 4 out of 12 pigs. These results suggest sub-optimal insulin production, possibly due to the adverse effects of CsA on the grafted β cells. The effect of CsA on adult porcine β cells was investigated and adverse effects were shown. Renal toxicity and adverse changes to the haematological parameters did not occur despite high CsA levels although minimal toxicity to the liver was observed. The results indicate that the use of CsA monotherapy in the maintenance phase to prevent rejection of allografted pancreatic β cells may become a subsequent problem over time. As an alternative to chronic immunossuppression, anti-CD3 monoclonal antibody was administered for 10 days in pigs. Using anti-CD3 alone, only 1 out 4 pigs showed cells positive for insulin. The addition of a 5-day CsA course administered the day before transplantation did not promote allograft survival. The use of DSG for 10 days with anti-CD3 promoted graft survival with the histology showing the 4 endocrine cells 3 weeks post-transplantation. An attempt was made to replace any form of immunossuppression by encapsulating fetal pig ICCs in barium alginate, which were able to remain viable when transplanted in NOD/SCID mice. Fibrosis was detected in BALB/c mice transplanted with encapsulated fetal ICCs suggesting that fetal pig ICCs shed antigens that elicit an immune response. Results from this study show that although fetal pig ICCs may be a viable source of insulin-producing cells, the use of CsA to prevent rejection has adverse effects on graft function. Encapsulation as well as transient immunosuppression is worthy of further investigation to prevent rejection of fetal pig ICCs.

Fetal pig islet-like cell clusters (ICCs) were transplanted into the thymus or omentum of STZ-induced diabetic pigs immunosuppressed with cyclosporine (CsA) and deoxyspergualin (DSG), as a potential treatment for type 1 diabetes. C-peptide levels in response to glucagon and arginine significantly increased over time using 1 litter of ICCs with highest levels obtained at 100 days post-transplantation. Increasing the number of ICCs to 2 litters was not advantageous. Histology of the graft showed all 4 pancreatic endocrine cells. Normoglycaemia was achieved for transient periods without insulin administration in 4 out of 12 pigs. These results suggest sub-optimal insulin production, possibly due to the adverse effects of CsA on the grafted β cells. The effect of CsA on adult porcine β cells was investigated and adverse effects were shown. Renal toxicity and adverse changes to the haematological parameters did not occur despite high CsA levels although minimal toxicity to the liver was observed. The results indicate that the use of CsA monotherapy in the maintenance phase to prevent rejection of allografted pancreatic β cells may become a subsequent problem over time. As an alternative to chronic immunossuppression, anti-CD3 monoclonal antibody was administered for 10 days in pigs. Using anti-CD3 alone, only 1 out 4 pigs showed cells positive for insulin. The addition of a 5-day CsA course administered the day before transplantation did not promote allograft survival. The use of DSG for 10 days with anti-CD3 promoted graft survival with the histology showing the 4 endocrine cells 3 weeks post-transplantation. An attempt was made to replace any form of immunossuppression by encapsulating fetal pig ICCs in barium alginate, which were able to remain viable when transplanted in NOD/SCID mice. Fibrosis was detected in BALB/c mice transplanted with encapsulated fetal ICCs suggesting that fetal pig ICCs shed antigens that elicit an immune response. Results from this study show that although fetal pig ICCs may be a viable source of insulin-producing cells, the use of CsA to prevent rejection has adverse effects on graft function. Encapsulation as well as transient immunosuppression is worthy of further investigation to prevent rejection of fetal pig ICCs.

No description available.

Lau Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abreviations --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Pancreas and its functions --- p.1 / Chapter 1.1.1 --- Structure of pancreas --- p.1 / Chapter 1.1.2 --- Exocrine function --- p.4 / Chapter 1.1.3 --- Endocrine function --- p.7 / Chapter 1.1.3.1 --- Pancreatic islet and islet cells --- p.7 / Chapter 1.1.3.2 --- Regulation of insulin secretion --- p.10 / Chapter 1.1.3.3 --- Mechanism for glucose-stimulated insulin release --- p.14 / Chapter 1.1.3.4 --- Bi-phase response of insulin secretion --- p.16 / Chapter 1.2 --- Pancreatic Renin-Angiotensin System --- p.19 / Chapter 1.2.1 --- Circulating RAS and local RAS --- p.19 / Chapter 1.2.2 --- RAS inhibitors --- p.25 / Chapter 1.2.2.1 --- Angiotensin converting enzyme inhibitor --- p.25 / Chapter 1.2.2.2 --- Non-specific Ang II receptor blocker --- p.28 / Chapter 1.2.2.3 --- Specific AT1 receptor antagonist --- p.29 / Chapter 1.2.2.4 --- Specific AT2 receptor antagonist --- p.30 / Chapter 1.2.3 --- RAS and Pancreas --- p.30 / Chapter 1.2.3.1 --- Expression and localization of pancreatic RAS --- p.30 / Chapter 1.2.3.2 --- Regulation of pancreatic RAS and its clinical relevance --- p.32 / Chapter 1.3 --- Islet Transplantation and RAS --- p.34 / Chapter 1.3.1 --- Whole pancreas and islet transplantation --- p.34 / Chapter 1.3.2 --- Problems encountered in islet transplantation --- p.36 / Chapter 1.3.3 --- Potential role of RAS in islet transplantation --- p.38 / Chapter 1.4 --- Diabetes Mellitus and RAS --- p.40 / Chapter 1.4.1 --- Diabetes Mellitus --- p.40 / Chapter 1.4.2 --- Type 1 diabetes and its animal model --- p.42 / Chapter 1.4.3 --- Type 2 diabetes and its animal model --- p.44 / Chapter 1.4.4 --- RAS blockade in diabetes patients --- p.46 / Chapter 1.4.5 --- Potential role of RAS in Diabetes Mellitus --- p.47 / Chapter 1.5 --- Aims of Study --- p.49 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Experimental animals and mouse models --- p.50 / Chapter 2.1.1 --- Experimental animals for islet isolation and transplantation --- p.50 / Chapter 2.1.2 --- Mouse model for type 2 diabetes --- p.51 / Chapter 2.2 --- Islet isolation and transplantation --- p.52 / Chapter 2.2.1 --- Enzymatic islet isolation --- p.52 / Chapter 2.2.2 --- Islet transplantation --- p.53 / Chapter 2.3 --- Biological assay on islet functions --- p.53 / Chapter 2.3.1 --- Measurement of islet insulin release --- p.53 / Chapter 2.3.2 --- Measurement of islet glucose oxidation rate --- p.56 / Chapter 2.3.3 --- Measurement of islet (pro)insulin biosynthesis --- p.59 / Chapter 2.3.4 --- Measurement of islet total protein synthesis --- p.60 / Chapter 2.4 --- Chronic losartan treatment --- p.62 / Chapter 2.5 --- Perfusion experiment of transplanted islet graft --- p.62 / Chapter 2.6 --- Insulin content of the islet graft --- p.63 / Chapter 2.7 --- Islet graft (pro)insulin and total protein biosynthesis --- p.64 / Chapter 2.8 --- Real-time RT-PCR Analysis --- p.64 / Chapter 2.8.1 --- Design of primers and probes --- p.67 / Chapter 2.8.2 --- Use of internal control --- p.69 / Chapter 2.8.3 --- RT-PCR reaction --- p.69 / Chapter 2.8.4 --- Calculation using the comparative CT method --- p.70 / Chapter 2.9 --- Western Blot Analysis --- p.71 / Chapter 2.10 --- Immunocytochemistry --- p.72 / Chapter 2.11 --- Statistical data analysis --- p.73 / Chapter Chapter 3 --- Results / Chapter 3 .1 --- Effect of Angiotensin II and Losartan on islet insulin release --- p.74 / Chapter 3.1.1 --- Insulin release from normal islets --- p.74 / Chapter 3.2 --- "Effect of Angiotensin II and Losartan on islet glucose oxidation rate, (pro)insulin and total protein biosynthesis" --- p.77 / Chapter 3.2.1 --- Glucose oxidation rate of isolated normal islets --- p.77 / Chapter 3.2.2 --- (pro)insulin and total protein biosynthesis of isolated normal islets --- p.77 / Chapter 3.3 --- Regulation of RAS components in islet transplantation --- p.81 / Chapter 3.3.1 --- Expression of RAS components in endogenous islets and transplanted islets --- p.81 / Chapter 3.3.2 --- Localization of AT1-receptor in endogenous islets --- p.87 / Chapter 3.3.3 --- Expression of AT1-receptor protein in endogenous and transplanted islets --- p.89 / Chapter 3.3.4 --- Relative abundance of RAS components in kidney and liver --- p.91 / Chapter 3.3.5 --- Insulin release from perfused transplanted islet graft --- p.93 / Chapter 3.3.5 --- (pro)insulin and total protein biosynthesis of transplanted islet graft --- p.96 / Chapter 3.4 --- Effect of Angiotensin II and losartan on diabetic islets --- p.99 / Chapter 3.4.1 --- Expression of RAS components in diabetic pancreas --- p.99 / Chapter 3.4.2 --- Localization of AT1 receptors in diabetic pancreas --- p.105 / Chapter 3.4.3 --- Insulin release from islets of type 2 diabetic mice --- p.107 / Chapter 3.4.4 --- (pro)insulin and total protein biosynthesis of islets from type 2 diabetic mice --- p.112 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Effect of angiotensin II and losartan on islet insulin release --- p.116 / Chapter 4.2 --- Existence of local RAS in pancreatic islets --- p.119 / Chapter 4.3 --- Regulation of islet RAS components in transplanted islets --- p.122 / Chapter 4.4 --- Clinical relevance of islet RAS in transplantation --- p.125 / Chapter 4.5 --- Regulation of islet RAS by type 2 diabetes --- p.126 / Chapter 4.6 --- Clinical relevance of islet RAS in type 2 diabetes --- p.134 / Chapter 4.7 --- Conclusion --- p.140 / Chapter 4.8 --- Further studies --- p.141 / Chapter Chapter 5 --- Bibliography --- p.142

Islet transplantation has emerged as a promising cell-based therapy for the treatment of diabetes, but its clinical efficacy remains limited by deleterious host responses that underlie islet destruction. In this dissertation, we describe the assembly of cell surface-supported thin films that confer molecular-level control over the composition and biophysicochemical properties of the islet surface with implications for improving islet engraftment. Specifically, the process of layer-by-layer (LbL) polymer self assembly was employed to generate nanothin films of diverse architecture with tunable properties directly on the extracellular surface of individual islets. Importantly, these studies are the first to report in vivo survival and function of nanoencapsulated cells, and have helped establish a conceptual framework for translating the diverse applications of LbL films to cellular interfaces. Additionally, through proper design of film constituents, coatings displaying ligands and bioorthogonally reactive handles may be generated, providing a modular strategy for incorporating exogenously derived regulators of host responses alongside native constituents of the islet surface. Towards this end, a strategy was developed to tether thrombomodulin to the islet surface in a site-specific manner, thereby facilitating local generation of the powerful anti-inflammatory agent, activated protein C. Collectively, this work offers novel biomolecular strategies for cell surface engineering with broad biomedical and biotechnological applications in cell-based therapeutics and beyond.