Background. Islet amyloid polypeptide (IAPP, also known as amylin) is a 37-amino acid peptide principally co-secreted with insulin from the beta-cells of the pancreatic islets. Some of the physiological actions of human amylin (hIAPP) include glucose regulation, suppression of appetite and stimulation of renal sodium and water reabsorption. Amylin deficiency and diminished post-prandial amylin response have been reported in advanced stages of type 1 and type 2 diabetes. In autopsy specimens of type 2 diabetes, amyloid is found in 40--90% of cases. During the characterization of islet morphology of aged hIAPP transgenic mice, I observed pathological features suggestive of immune dysregulation. Review of literature also suggested possible immuno-modulating functions of human amylin in in vitro experiments. Since autoimmunity and innate immunity are implicated in aging and diabetes, I explored the immunological role of amylin with particular focus on CD4+CD25+ T regulatory cells and toll-like receptors (TLR) which are known mediators of autoimmunity and innate immunity respectively. / Conclusions. Human amylin may play an important role in modulating immunity mainly through stimulating CD4+CD25+ Treg cells, decreasing PLN and altering expression of TLR-4 and cytokines. If these findings are confirmed in in vivo model, human amylin has the potential to become a novel and promising therapy to prevent and reverse autoimmune disease such as autoimmune type 1 diabetes. / Hypothesis. Human amylin may have immunomodulating effects which may have implications on pathogenesis of autoimmune type 1 diabetes. / Materials and methods. Male hemizygous hIAPP transgenic mice (n=32) and their nontransgenic littermates (n=20) were fed with normal chow and studied longitudinally up to 18 months of age with measurement of plasma insulin, glucose and amylin at regular intervals. Detailed oral glucose tolerance test, intra-peritoneal insulin tolerance test, insulin and amylin protein expression were examined at 3, 7, 12 and 18 months of age. Histological changes of pancreas and spleen including changes in CD4+CD25+ T regulatory cells and cytokines were examined at 12 and 18 months. / Objectives. (1) I systemically characterized the morphological, functional and immune regulatory role of human amylin in aged hIAPP transgenic mice which include metabolic profiles, plasma levels of amylin and insulin as well as morphological changes of pancreatic lymph nodes (PLN). (2) I then examined splenic expression of TLR-4 associated changes in cytokines (TNF-alpha, TGF-beta, and IL-6). (3) I also examined the expression level of receptor activity modifying proteins (RAMPs) in pancreas and spleen. (4) I finished by investigating the role of human amylin on stimulating CD4+CD25+ T regulatory (Treg) cells in hIAPP transgenic mice and peripheral blood monocytes (PBMC) from healthy subjects. / Results. (1) With aging, the hIAPP transgenic mice demonstrated increased plasma amylin, decreased plasma insulin, reduced insulin to amylin ratio and improved insulin sensitivity (p<0.05). (2) The aged hIAPP transgenic mice showed changes in immune function as indicated by: (a) Reduced number and size of PLN (p<0.05). (b) Decreased expression level of TLR-4 in splenocytes (p<0.05). (c) Increased expression of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) protein but decreased level of IL-6 in splenocytes (p<0.05). (3) The changes in the levels of immune cytokines such as IL-1, IL-2, IL-4, IL-10, IL-17, interferon-gamma and GM-CSF were similar between hIAPP transgenic and nontransgenic mice (p>0.05). (4) The levels of RAMP1, RAMP2, and RAMP3 were higher in the spleen of hIAPP transgenic mice than nontransgenic mice (p<0.05). (5) The hIAPP transgenic mice showed higher percentage of CD4+CD25+ Treg cells compared with nontransgenic littermates. Treatment with human amylin, but not rat amylin, increased the percentage of FoxP3+CD4+CD25+ Treg cells in both splenic T lymphocytes of hIAPP transgenic mice and PBMCs of healthy subjects ex vivo (p<0.05). / He, Lan / Adviser: Juliana C.N. Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 176-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI) / Type I diabetes is an autoimmune disorder characterized by the loss of insulin producing islet cell mass. While daily insulin injection provides an easy means of glycemic control, it does not prevent long-term complications associated with diabetes. Islet transplantation has been suggested as a permanent cure for type 1 diabetes. However, the recurrence of host immunity and shortage of donor islets hinder the prevalence of islet transplantation. Biomaterial strategies provide an alternative route to solving the problems associated with host immune response and shortage of donor islets. One highly recognized platform for achieving these goals are hydrogels, which are hydrophilic crosslinked polymers with tissue-like elasticity and high permeability. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, including encapsulation of pancreatic islets and serving as a material platform for pseudo-islet differentiation. PEG hydrogels formed by mild and rapid thiol-ene photo-click reactions are particularly useful for studying cell behaviors in three-dimension (3D). Thiol-ene PEG-based hydrogels can be rendered biodegradable if appropriate macromer and cross-linker chemistry is employed. However, the influence of hydrogel matrix properties on the survival, growth, and morphogenesis of cells in 3D has not been fully evaluated. This thesis aims at using norbornene-functionalized PEG macromers to prepare thiol-ene hydrogels with various stiffness and degradability, from which to study the influence of hydrogel properties on pancreatic cell fate processes in 3D. Toward establishing an adaptable hydrogel platform for pancreatic tissue engineering, this thesis systematically studies the influence of hydrogel properties on encapsulated endocrine cells (e.g., MIN6 beta-cells) and exocrine cells (PANC-1 cells), as well as human mesenchymal stem cells (hMSC). It was found that thiol-ene photo-click hydrogels provide a cytocompatible environment for 3D culture of these cells. However, cell viability was negatively affected in hydrogels with higher cross-linking density. In contrast to a monolayer when cultured on a 2D surface, cells with epithelial characteristic formed clusters and cells with mesenchymal features retained single cell morphology in 3D. Although cells survived in all hydrogel formulations studied, the degree of proliferation, and the size and morphology of cell clusters formed in 3D were significantly influenced by hydrogel matrix compositions. For example: encapsulating cells in hydrogels formed by hydrolytically degradable macromer positively influenced cell survival indicated by increased proliferation. In addition, when cells were encapsulated in thiol-ene gels lacking cell-adhesive motifs, hydrolytic gel degradation promoted their survival and proliferation. Further, adjusting peptide crosslinker type and immobilized ECM-mimetic bioactive cues provide control over cell fate by determining whether observed cellular morphogenesis is cell-mediated or matrix-controlled. These fundamental studies have established PEG-peptide hydrogels formed by thiol-ene photo-click reaction as a suitable platform for pancreatic tissue engineering