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.

Free radicals are thought to be involved in the destructive process of beta cells in Type 1 diabetes mellitus. Studies were performed to test the hypotheses (1) that malondialdehyde (MDA), a by-product of lipid peroxidation, affects $ beta$-cell function and integrity in vitro and (2) that such effects might be prevented in the BB rat (a model of spontaneous autoimmune diabetes) in vivo by administration of $ alpha$-phenyl-N-tert-butylnitrone (PBN), a free radical spin trap. First, islets of Wistar-Furth rats were studied at 12, 24 and 40 hr of culture in either 5.5, 11 or 16.5 mM glucose, and MDA at a range of concentrations ($6 times10 sp{-12}$-10$ sp{-3}$M). High concentrations of MDA inhibited glucose-stimulated insulin release without corresponding decreases in islet insulin content, suggesting that in situations with high predicted islet free radical content (e.g., autoimmune insulitis) beta cell function may be affected even before the cells are destroyed. Second, 28 diabetes-prone (BBdp) and 13 non diabetes-prone (BBn) rats were given PBN (20 mg/kg) s.c. 2x/day and 27 BBdp and 12 BBn rats received an equal volume of saline. PBN was able to decrease MDA in the absence of the autoimmune process and is remarkably non-toxic. However, it did not prevent diabetes for reasons which may include its concentration at the site of the inflammatory process or specificity to types of radicals trapped. Because it did decrease MDA, either a higher dose or a combination of PBN with other agents may hold promise for disease prevention.

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.

Typescript / Copies of two published papers by the author, in back / Bibliography: leaves 111-133 / 133 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1986

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Thesis (Ph.D.)--Baylor University, 2009. / Includes bibliographical references (p. 126-143).

No description available.

No description available.

Orientador: Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T07:06:58Z (GMT). No. of bitstreams: 1 Arantes_VanessaCristina_D.pdf: 1696436 bytes, checksum: 2db258b560bf7ee0ec64de7441adfe76 (MD5) Previous issue date: 2006 / Resumo: Em animais, a desnutrição intra-uterina exerce efeitos marcantes sobre o desenvolvimento fetal e pós-natal. Sabe-se que animais desnutridos apresentam níveis elevados de ácidos graxos plasmáticos e esses, por sua vez, são responsáveis por alterar a secreção de insulina. Neste trabalho, verificamos a expressão do fator de transcrição PDX-1, da p38/SAPK2, o metabolismo da glicose e a secreção de insulina em ilhotas de ratos mantidos durante o período fetal e da lactação com uma dieta normoprotéica (17% de proteína) ou hipoprotéica (6% de proteína). Cultivamos as ilhotas por 48 horas em meio de cultura contendo 5.6 mM/L de glicose, na ausência ou presença de0.6 mM/L de ácido palmítico. A secreção de insulina em ilhotas isoladas em resposta 16,7 mmol/L de glicose foi reduzida em ratos desnutridos, no entanto, quando na presença de ácido graxo, observou-se um aumento. Em 2.8 mmol glicose/L,houve diminuição do metabolismo da glicose em ilhotas de desnutridos .Entretanto, quando estimuladas com 16.7 mmol/L de glicose, tanto as ilhotas de desnutridos como as do controle, apresentaram acentuada redução na oxidação da glicose, na presença de ácido graxo. Os níveis de mRNA do PDX-1 e da insulina aumentaram significativamente quando na presença de ácido graxo em ambos os grupos. O efeito do ácido palmítico sobre a expressão protéica de PDX-1 e da p38/SAPK2 apresentou-se similar em ambos os grupos, mas o aumento foi muito mais evidente em ilhotas de desnutridos. Esses resultados demonstram a complexa relação entre nutrientes no controle da secreção de insulina e mostram queos ácidos graxos desempenham um papel importante na homeostasia da glicose, por afetar mecanismos moleculares e as vias de acoplamentsecreção de insulina / Abstract: A severe reduction in insulin release in response to glucose is consistently noticed in protein-deprived rats and is attributed partly to the chronic exposure to elevated levels of free fatty acids. Since the pancreatic and duodenal transcription factor homeobox 1 (PDX-1) is important for the maintenance of B-cell physiology, and since PDX-1 expression is altered in the islets of rats fed a low protein diet, we assessed PDX-1 and insulin mRNA expression, as well as PDX-1 and p38/SAPK2 protein expression, in islets from young rats fed low (6%; LP) or normal (17%; C) protein diets and maintained for 48 h in culture medium containing 5.6 mmol glucose/L with or without 0.6 mmol palmitic acid/L. We also measured glucoseinduced insulin secretion and glucose metabolism. Insulin secretion by isolated islets in response to 16.7 mmol glucose/L was reduced in LP compared to C rats. In the presence of free fatty acids, there was an increase in insulin secretion in both groups At 2.8 mmol glucose/L, the metabolism of this sugar was reduced in LP islets, regardless of the presence of this fatty acid. However, when challenged with 16.7 mmol glucose/L, LP and C islets showed a severe reduction in glucose oxidation in the presence of free fatty acid. The PDX-1 and insulin mRNA were significantly higher when free fatty acid was added to the culture medium in both groups of islets.The effect of palmitic acid on PDX-1 and p38/SAPK2 protein levels was similar in LP and C islets, but the increase was much more evident in LP islets. These results demonstrate the complex interrelationship between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis by affecting molecular mechanisms and stimulus/secretion coupling pathways / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular