O Diabetes mellitus tipo 1 (DM1), na maioria dos casos, é causado pela destruição de células β pancreáticas, levando à hiperglicemia. Atualmente a única fonte de novas células β e os únicos tratamentos capazes de restaurar o padrão fisiológico de secreção de insulina nesses pacientes são o transplante de pâncreas e de ilhotas pancreáticas. O transplante de ilhotas apresenta problemas relacionados a enxertia, devido principalmente a baixa vascularização, o que leva à morte de células β nos primeiros dias pós-transplante. Células-tronco mesenquimais apresentam características interessantes para o tratamento do DM1. A primeira aplicação explorada nesse trabalho foi a capacidade de diferenciação de MSCs humanas e murinas em células produtoras de insulina (CPIs). A identidade das células isoladas foi confirmada pela caracterização imunofenotípica e pela capacidade de diferenciação adipogênica e osteogênica in vitro. Quatro protocolos de diferenciação em CPIs foram testados em MSCs derivadas de ilhotas pancreáticas e um em MSCs derivadas de rim murino. A análise da expressão gênica de insulina em células diferenciadas em todos os protocolos testados mostrou níveis insignificantes ou nulos de expressão desse hormônio. A segunda aplicação explorou o co-transplante de ilhotas pancreáticas com MSCs derivadas de rim em camundongos diabéticos. Os resultados mostraram aumento da taxa de cura e melhora na glicemia pós-transplante, bem como uma tendência ao aumento do conteúdo total de insulina em animais co-transplantados em comparação com animais que receberam apenas ilhotas. Não houve diferenças no peso e teste de tolerância à glicose entre os grupos. Foi observado aumento na vascularização do enxerto nos animais que receberam MSCs. Paralelamente, foi estudada a associação de variantes alélicas dos genes PTPN22, KIR, HLA classe I e II e a susceptibilidade ao desenvolvimento de DM1 em uma população do Rio Grande do Sul. Foi observada associação entre o alelo 1858T e o risco aumentado de DM1. A genotipagem do KIR e HLA-C mostrou uma frequência maior de alelos do grupo 2 do HLA-C em controles não diabéticos, bem como o genótipo 2DL1/C2+, sugerindo um papel protetor desse genótipo. Além disso, indivíduos com haplótipo KIR2DL2/DR3+ e KIR2DL2/DR3/DR4+ tem risco aumentado de desenvolvimento de DM1. MSCs parecem possuir baixa capacidade de diferenciação em células β in vitro, entretanto, possuem efeitos benéficos importantes quando cotransplantadas com ilhotas pancreáticas. Essa aplicação tem grande potencial e deveria ser testada em estudos clínicos com o objetivo de melhorar a enxertia e diminuir o número de ilhotas necessárias para cada paciente. / Type 1 Diabetes (DM1), in almost all the cases, is caused by the destruction of beta-cells by cells of the immune system, leading to hyperglycemia. The only source for new beta-cells available is through the pancreas and islet transplantation, two treatments able to restore insulin secretion pattern in this patients. Islet transplantation presents issues related to grafting, caused mainly by poor vascularisation post-transplant, leading to betacell death in the first days after transplantation. Mesenchymal stem cells have interesting characteristics to the treatment of DM1. The first application explored in this work was testing the capacity of differentiation of human and mouse MSCs into insulin-producing cells (CPIs). Identity of isolated cells was confirmed by immunophenotyping and potential of adipogenic and osteogenic differentiation in vitro. Four protocols were tested in human islet-derived MSCs and one in mouse kidney-derived MSCs to generate CPIs. Analysis of insulin expression in differentiated cells from all protocols showed no or very little expression levels of this hormone. The second application was to evaluate the role of MSCs in the co-transplantation with pancreatic islets in diabetes mice. Our results showed an increased number of cured mice and a decrease in glycemic levels post transplant in islet+MSCs group, as well as a tendency to an increase in total insulin content in islet+MSCs compared with islet-only group. No differences could be found in weight and intraperitoneal glucose tolerance test between groups. An increase in graft vascularisation was observed in MSCs-receiving animals. At the same time, we studied the association of allelic variants in PTPN22, KIR, HLA class I and II genes and its association with the developing of DM1. We reported and association of the 1858T allele and an increased risk of DM1. Genotyping shows an increased frequency of group 2 alleles (C2) of HLA-C in controls as well as the 2DL1/C2+ genotype, suggesting a protective role of this genotype. Moreover, individuals with KIR2DL2/DR3+ and KIR2DL2/DR3/DR4+ haplotypes have increased risk of developing DM1. MSCs seem to have low capacity of in vitro differentiation in a beta-cell phenotype, however, they exert important benefic effects when co-transplanted with pancreatic islets in diabetic mice. This application has great potential and should be tested in clinical trials aiming the improvement of islet grafting and decrease in the number of islets needed for transplantation.

Type 1 diabetes is an autoimmune disease in which patients' insulin-secreting beta cells in pancreatic islets are destroyed by their own immune system, leading to unregulated blood glucose levels and severe complications. Its only treatment is intensive insulin therapy, which carries the risk of hypoglycemic episodes and can result in seizures, coma, and even death. Islet transplantation has recently become an alternative, albeit experimental, treatment for type 1 diabetes patients. More than one donor graft is usually required to render recipients insulin independent, making the shortage of donor tissue an extremely important challenge in islet transplantation. Identifying the cell type that has the ability to differentiate into islet-like tissue is an important area of study. In this study, I hypothesized that insulin secreting human islet-like clusters could be generated from pancreatic ductal cells, a potential pancreatic progenitor cell type. Islet-like clusters were generated using crude exocrine tissue from human cadaveric donors. This crude exocrine tissue contained a large number of ductal cells, as well as other pancreatic cell types. To evaluate insulin secretion by human islet-like clusters, a static incubation system was set up and tested using Min6 cells, a known insulin-secreting cell line. Using static incubation, significant increases in insulin secretion by islet-like clusters were observed when the clusters were exposed to higher glucose levels and GLP-1, a known insulin secretagogue. Presence of corresponding C-peptide secretion demonstrated that de novo insulin secretion occurred. Furthermore, basal insulin secretion increased as culture stages progressed. An attempt was made to generate islet-like clusters using ductal cells purified by fluorescent activated cell sorting or magnetic activated cell sorting. Nevertheless, it was difficult to ensure survival and proliferation of purified ductal cells. Further studies will be necessary to confirm the role of ductal cells in the generation of islet-like clusters using the crude exocrine tissue, as well as to identify factors that can promote ductal cells proliferation after cell sorting. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

No description available.

Despite the considerable expansion of knowledge in the development of pancreatic cancer, there has been little progress made in facilitating an early diagnosis of this disease and predicting an accurate response to treatment. We aim to translate this knowledge to clinical practice by using a prospective database of precursor cystic lesions in pancreas cancer, assessing the use of over-expressed genes in pancreatic juice as a surrogate marker of these pancreas cancer and finally, downstream of these changes at the genetic level, use metabolomic techniques to look for biomarkers in pancreas cancer in serum. In the first study, we investigate the natural history of pancreatic cystic neoplasms, specifically IPMNs, using a prospectively collected database to examine the profiles and outcomes of main duct IPMN, branch duct IPMN and cystic lesions measuring less than 3 cm in size. A total of 99 patients with suspected pancreatic cystic tumours were enrolled over 3 years. Median follow-up was 24 months (range 0 – 124). Cystic tumours comprised of 13 MD-IPMN, 40 BD-IPMN, 11 MCN and 8 adenocarcinomas among others. The complete cohort showed an overall risk of adenocarcinoma of 8%. Main duct IPMN showed a cumulative risk of 46% with evidence of progression of disease in a further 23%. The associated mortality in MD-IPMN was related to the underlying adenocarcinoma and was 38% in our group. The incidence of adenocarcinoma in branch duct IPMN was 11% with disease progression seen 13.8%. Evidence of extra-pancreatic malignancies was seen in 37.7% of patients with IPMN. In the second study, we explore the feasibility of gene expression profiling from RNA isolated from matched pancreatic juice and tumour tissue in patients with pancreatic cancer and pancreatic cystic tumours. RNA was isolated and Poly(A) PCR was used to globally amplify the RNA. RT-PCR was used to measure expression levels of 18 genes common to both pancreas cancer and pancreatic cystic tumours. Spearman’s rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. One gene out of eighteen, MSLN (p<0.008), showed significant correlation in the expression levels between paired pancreatic juice and tissue samples in pancreas cancer. In the cystic tumour group, only one gene MMP-7 (p<0.01), showed a significant correlation between paired juice and tissue samples. When the whole cohort was analysed for the false discovery rate, these genes did not exhibit statistically significant correlation between the samples. RNA analysis of pancreatic juice is feasible using the Poly(A) cDNA technique and correlation of gene expression is shown to exist, albeit with low sensitivity, indicating its potential use in clinical practice with small tissue and juice samples. In the final study, we performed a literature review on the use of metabolomics in pancreas cancer. We performed metabolic profiling of serum samples from selected cancer patients and noncancerous controls using UPHLC-MS to generate and compare the metabolic profiles in serum samples from a cohort of patients with pancreas cancer, ampullary cancer and endocrine cancer. Thirty nine serum samples (including 19 pancreatic cancers, 9 ampullary cancers and 5 endocrine cancers) and 21 matched HUSERMET controls were analysed using Ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) in both positive and negative ESI modes. The output was generated as a data matrix of mass spectral features with related accurate m/z and retention time pairs. The data was then signal corrected and individual peaks were normalised and the resultant spectra were compared against a metabolite reference library and analysed using univariate and multivariate statistical tests. We found a disparity in the metabolite peaks between the cases and controls on PCA that did not permit the accurate interpretation of the data in the case study set compared to the control set. No obvious reason other than metabolite degradation during storage could account for this difference. PC-DFA analysis of metabolite peaks between pancreas cancer, ampullary cancer and endocrine cancer showed significant difference between endocrine cancers and the other two groups. Significant ESI positive metabolites included those involved in lipid pathways and metabolites involved in glucose metabolism. There is encouraging scope for studies using prospective controls to identify and develop metabolic biomarkers in pancreas cancer.

Orientador : Arrigo Antonio Raia / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-07-18T01:51:50Z (GMT). No. of bitstreams: 1 Leonardi_LuizSergio_D.pdf: 2318868 bytes, checksum: c3dff364d3036b7fa9919674a187e939 (MD5) Previous issue date: 1972 / Resumo: Não informado. / Abstract: Not informed. / Doutorado / Doutor em Ciências Médicas

Orientador: Maria Juana Escribano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-19T11:55:33Z (GMT). No. of bitstreams: 1 Garcia_IrandaiaUbirajara_D.pdf: 9327229 bytes, checksum: 1adb737cb20a365e67d01b2e765f4679 (MD5) Previous issue date: 1994 / Resumo: A carcinogênese experimental em hamster, induzida por nitrosaminas, é um processo multifásico dando origem a adenocarcinomas na fase terminal, semelhante à doença humana. A resposta humoral e celular neste modelo, levou aos seguintes resultados: Quando os tecidos são tratados com antissoro específico aos linfócitos "T" de hamster, observamos uma migração de células "T" através do tecido exócrino em paralelo a neo-vascularização intensa (angiogênese). Esta migração foi observada precocemente, antes que pudesse constatar lesões nas análises por histologia convencional. Parece que as células em possível cooperação com outras células imunocompetentes, foram capazes de atacar ou destruir as acinares sem alterações morfológicas evidentes, ao lado de canais hiperplásicos correspondentes às lesões pré-neoplásicas. Entretanto, não foi observado linfócitos "T" em contacto direto com os canais em hiperplasia epitelial. Ao longo da carcinogênese observamos um aumento progressivo da síntese de imunoglobulinas circulantes. Esta foi medida pelo método semi-quantitativo "dot-blot", utilizando antissoro anti-lgG total de hamster. O método da imunoperoxidase indireta nos cortes histológicos, mostrou a presença de auto-anticorpos dirigidos provavelmente contra neoantígenos , que surgiram em consequência das lesões induzidas. ... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Experimental carcinogenesis in hamster, induced by nitrosamines, is a multiphasic processus ultimatly leading to adenocarcinomas closely ressembling the human illness.A follow-up of hUl11oral and cellular immuncrcsponscs in this modcl allowcd thc following results: When tissues were processed with an antiserum specifc for hamster T-lymphocytes, we observed a migration of T-cells througth the exocrine tissue concomitant with abundant neovascularitation (angiogenis). Migration occurred far before lesiolls could be Iloticed by conventional histology. Apparently, these cells, in possible cooperatin with other immunocompetent cells, were able to attack or destroy acini with no signifíant morphological alterations as well as hyperplasic ducts which are preneoplasic lesions. T-Iynphocytes in neoplasic ducts were, on the contraty, not observed. Along the carcinogenesis there also was an increase in immunoglobuline synthesis in blood-circulation This was evaluated by semiquantitative nitrocellulose immuno "dot blot" using anti-whole Ig antiserum. Indirect immunoperoxidase in the tissue sections showed presence of autoantibodies directed against neoantigens appearing as a consequence of theinduced lesions. ... Note: The complete abstract is available with the full electronic digital thesis or dissertations / Doutorado / Doutor em Imunologia

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13267号 / 論医博第2181号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 篠原 隆司, 教授 影山 龍一郎 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Statins are widely used in the treatment of atherosclerosis and hypercholesterolemia, both of which are comorbidities of obesity. However, the effects statins have on insulin homeostasis are relatively unknown and may increase one’s risk for type-II diabetes mellitus. INS-1 pancreatic β-cells, were cultured in 11 mM glucose with 25, 50, 100, 200 nM statin or without. Specifically, this study observed the effects that pitavastatin, simvastatin, lovastatin, and pravastatin have on insulin secretion, insulin content, and ROS levels. GSIS was measured after statin and non-statin exposed cells were incubated in 12 mM glucose KRB. Insulin content was measured after trypsinization and subsequent lysing of cells. Both were analyzed via FRET based HTRF assay. ROS levels within cells were measured following statin exposure during a 2-hour period of 12 mM glucose oxidation after DCF was added. Analysis was done using a Tecan™ fluorescent microplate reader. Pitavastatin, simvastatin, and lovastatin decrease glucose stimulated insulin secretion and insulin content as compared to control. All concentrations of pitavastatin reduced insulin secretion proportionally to insulin content, suggesting it does so through impairment of insulin synthesis or storage. Simvastatin reduced insulin secretion and content in a dose dependent manner, however when secretion was adjusted for % content, data showed that high doses of simvastatin reduced insulin content in a greater fashion than insulin secretion, suggesting both secretory mechanisms and storage/synthesis were impaired. Lovastatin reduced insulin secretion by a greater amount than its reduction of insulin content, suggesting that it impaired insulin secretion via secretory mechanism impairment. Pravastatin did not have an effect on either insulin secretion or insulin content at any concentration. Cells were also tested to determine if pitavastatin, simvastatin, or lovastatin induced a change in ROS levels within the cell. None of the three statins tested caused a statistically significant change in ROS levels at all concentrations. These results suggest that pitavastatin, simvastatin, and lovastatin may impair insulin secretion in patients with high blood glucose. As such, clinical guidelines for statin therapy use in those who are at risk, or suffer from, diabetes may need to be reevaluated.

No description available.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).