Thesis (M.D.)—Boston University

A method was developed to produce and incorporate a network of discrete, genipin-crosslinked gelatin microfibers around a pancreatic islet within a barium alginate microcapsule. This technique allows for the encapsulation of a porous fibrous matrix without the geometrical restrictions required for cellular aggregate seeding. Microfibers were produced from a novel vortex-drawn extrusion system with an alginate support matrix. Optimization culminated in a hydrated fiber diameter of 22.3 ± 0.4 μm, a 98% reduction in cross sectional area, while making the process more reliable and less labour intensive. The optimized microfibers were encapsulated at 40 vol% within 294 ± 4 μm 1.6% barium alginate microparticles by an electrostatic-mediated dropwise extrusion system. Pancreatic islets extracted from Sprague Dawley rats were encapsulated within the microparticles, and analyzed over a 21-day preliminary in vitro study. Acridine orange and propidium iodide fluorescent viability staining and light microscopy indicated a significant increase in viability for the fiber-laden particles relative to fiber-free control particles at days 7, 14, and 21. The fiber-laden system also reduced the incidence of disrupted islet cohesion from 31% to 8% at day 21, and showed evidence of islet-fiber adhesion. Preliminary investigations into insulin secretion and metabolic activity showed no significant difference between test and control groups. Further investigation into benefits of islet encapsulation within an extracellular matrix fiber network will be the subject of future studies with this body of work serving as a foundation. The system developed in this investigation could be developed into a modular scaffold system for tissue engineering beyond the field of islet research. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-08-18 15:05:50.917

Acredita-se que a diminuição progressiva da massa de células observada durante a evolução do diabetes mellitus tipo 2 (DM 2) ocorra por apoptose deste tipo celular. As tiazolidinedionas (TZDs), uma classe de medicamentos utilizada no tratamento do DM 2, atuam como ligantes dos receptores ativados por proliferadores de peroxissomos (PPAR) e e promovem diminuição da resistência periférica à insulina. Embora existam estudos controversos, tem se especulado que as TZDs possam exercer efeitos diretos sobre as células pancreáticas, prevenindo a perda por apoptose e melhorando a sua viabilidade. O objetivo deste estudo foi avaliar os efeitos diretos da Pioglitazona (PIO) na concentração de 10 M sobre a viabilidade funcional e o índice de apoptose de ilhotas pancreáticas isoladas de ratos Wistar expostas a concentrações fisiológica (5,6 mM) e suprafisiológica (23 mM) de glicose durante 24, 48 e 72 horas. A viabilidade funcional foi avaliada pela análise da secreção de insulina estimulada por glicose e do conteúdo total de insulina nas ilhotas. O índice de apoptose foi avaliado pela medida da fragmentação do DNA, da expressão do RNAm dos genes Bcl2 (anti-apoptótico) e Bax (pró-apoptótico) e da atividade proteolítica da caspase-3 em ilhotas tratadas e não tratadas com a PIO. Em 5,6 mM de glicose, não se observou efeito significativo sobre a secreção de insulina, mas a avaliação do conteúdo total de insulina evidenciou uma diminuição transitória nas ilhotas tratadas com PIO por 24 horas, seguida por um aumento no conteúdo de insulina quando as ilhotas foram cultivadas por 48 e 72 horas em presença da droga. Em relação à avaliação da apoptose, observou-se uma diminuição na expressão do RNAm do gene Bax nas ilhotas tratadas com PIO por 24 horas, entretanto, após 48 e 72 horas, houve um aumento da expressão do RNAm deste gene nas ilhotas tratadas com a droga. Não foram observadas diferenças estatisticamente significativas na expressão do RNAm do gene Bcl2 em nenhum dos tempos estudados e a avaliação da apoptose determinada pela medida da fragmentação do DNA somente demonstrou uma diminuição do índice de apoptose após 48 horas de tratamento com a PIO. Em 23 mM de glicose, a PIO promoveu um aumento transitório na secreção de insulina estimulada por glicose e no conteúdo total de insulina (após 48 horas), no entanto, após 72 horas, observou-se diminuição significativa no conteúdo total de insulina. Em relação à apoptose, o tratamento com PIO determinou um aumento do índice de apoptose medido pela fragmentação do DNA e da atividade proteolítica da caspase-3 após 48 e 72 horas e uma diminuição da expressão do RNAm do gene Bcl2 nos tempos 24 e 48 horas. Os resultados do presente estudo sugerem que os efeitos diretos da PIO sobre as ilhotas pancreáticas murídeas em cultura variam de acordo com a concentração de glicose a qual as ilhotas estão expostas: em concentração fisiológica de glicose, a PIO parece exercer efeitos diretos benéficos, enquanto em concentração suprafisiológica de glicose, ela exerce efeitos diretos deletérios sobre a viabilidade funcional e o índice de apoptose de ilhotas pancreáticas murídeas em cultura. / The progressive decrease in -cell mass observed during the evolution of type 2 diabetes (T2DM) is believed to occur due to cell apoptosis. Thiazolidinediones (TZDs), a class of agents used for the treatment T2DM, act as ligands of the peroxisome proliferator-activated receptor (PPAR) and and decrease peripheral insulin resistance. Although still controversial, some studies have shown a direct effect of TZDs on pancreatic -cell, preventing cell loss due to apoptosis and improving their viability. The objective of this study was to evaluate the direct effects of 10 M Pioglitazone (PIO) on functional viability and apoptosis rate of islets isolated from Wistar rats exposed to physiological (5.6 mM) and supraphysiological (23 mM) glucose concentrations during 24, 48 and 72 hours. The functional viability was evaluated by the analysis of insulin secretion after glucose challenge and of islet total insulin content. Apoptosis rate was evaluated by measurement of DNA fragmentation, of Bcl2 (antiapoptotic) and Bax (proapoptotic) mRNA expression and of proteolytic activity of caspase-3 in pancreatic islets treated or not with PIO. At 5.6 mM glucose concentration, no significant effects in insulin secretion were observed, while a transitory decrease (after 24 hours) followed by an increase in total insulin content was observed in islets treated with PIO for 48 and 72 hours. Regarding apoptosis, a lower expression of Bax mRNA was detected in islets treated with PIO for 24 hours, followed, however, by an increase in the expression of this gene after 48 and 72 hours of drug exposition. PIO treatment did not promote significant changes in Bcl2 mRNA expression, while decreased the apoptosis rate measured by DNA fragmentation only after 48 hours of exposition. At 23 mM glucose concentration, PIO treatment elicited a transitory increase in insulin secretion after glucose challenge and in islet total insulin content after 48 hours followed by a decrease in the islet total insulin content after 72 hours. Concerning apoptosis, PIO treatment determined an increase in the apoptose rate measured by DNA fragmentation and by proteolytic activity of caspase-3 after 48 and 72 hours and a decrease in Bcl2 mRNA expression after 24 and 48 hours. These findings suggest that the direct effects of PIO on pancreatic islets depend on glucose concentration to which they are exposed: while under physiological glucose concentration the direct effects seem to be beneficial, under supraphysiological glucose concentration, PIO exerts direct deleterious effects on the functional viability and on the apoptosis rate of murine pancreatic islets.

Não é claro como o receptor de glicocorticóide (GR) contrarregula a atividade do STAT5 na transição da gestação para a lactação. Dexras1 é uma pequena proteína G ativada por dexametasona (DEX), que regula morfologia celular, crescimento, etc. Neste estudo detectamos a expressão de Dexras1 em células beta de ilhotas pancreáticas. DEX induz a expressão de Dexras1 em células RINm5F, que está aumentada na gestação e diminuída na lactação. A expressão protéica de 11<font face=\"Symbol\">&#946HSD1, enzima ativadora de glicocorticóides (GCs), segue esse perfil. A ligação tanto do GR como do STAT5, analisada por ChIP assay, ao promotor do gene da Dexras1 aumentada por DEX é revertida por prolactina (PRL), e está diminuída e aumentada na gestação e lactação, respectivamente. DEX induz a associação ao GR, fosforilação e translocação nuclear do STAT5b. O silenciamento gênico de Dexras1 promoveu aumento da secreção de insulina, e aumentou os níveis de pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 e PKA. Sendo assim, a regulação de Dexras1 por PRL e GCs contribui para a secreção de insulina característica do periparto. / It is not clear how glucocorticoid receptor (GR) counteracts STAT5 activity during the transition of pregnancy to lactation. Dexras1 is a small G protein activated by dexamethasone (DEX) that controls cell morphology, growth, etc. In the present study we detected Dexras1 expression in pancreatic beta cell. DEX induces Dexras1 expression in RINm5F cells, which is increased in pregnancy and decreased in lactation. The expression of 11<font face=\"Symbol\">&#946HSD1, the glucocorticoids (GCs) activating enzyme, followed Dexras1 profile in pancreatic islet. Both GR and STAT5b bindings to Dexras1 gene promoter, analyzed by ChIP assay, are increased by DEX and PRL counteracts this effect. Both bindings are decreased in pregnancy and increased in lactation. DEX induces STAT5b association to GR, phosphorylation and nuclear translocation. Dexras1 knockdown using small interference RNA (si-RNA) promoted an increase in insulin secretion, as well as increased levels of pERK1/2, pCREB, pPKC<font face=\"Symbol\">&#948 and PKA. Thus, Dexras1 regulation by PRL and GCs contributes to insulin secretion during peripartum.

Made available in DSpace on 2017-07-21T19:59:57Z (GMT). No. of bitstreams: 1 Nayara Carvalho Leite.pdf: 2701096 bytes, checksum: 1dca8ecc3a7945789cec9125b5a0c33b (MD5) Previous issue date: 2013-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A obesidade é um fator de risco para o desenvolvimento do diabetes tipo 2. O exercício físico reduz o tecido adiposo, modula a secreção e ação da insulina preservando a homeostase glicêmica. A administração de glutamato monossódico (MSG) induz lesões hipotalâmicas que levam a obesidade. O controle da secreção de insulina depende da formação do ATP nas células β pancreáticas, processo acoplado a rotas metabólicas glicolíticas e mitocondriais. O presente estudo investigou o efeito do exercício físico no metabolismo da glicose em ilhotas pancreáticas de ratos MSG-obesos. A obesidade foi induzida pela administração de MSG (4g/Kg). Controles (CON) receberam salina. Aos 21 dias os animais foram separados em 4 grupos CON-SED (sedentários); CON-EXE (exercitados); MSG-SED e MSG-EXE. O exercício consistiu em natação (3x/semana/30min). As ilhotas foram isoladas e incubadas com glicose (16,7 mM) na presença ou ausência dos seguintes bloqueadores do metabolismo da glicose: 1-Ácido Iodoacético (IAA, 1mM), bloqueia a glicólise; 2– Ácido alfa-ciano-4-hidroxicinâmico (-CHC, 1mM), evita o metabolismo do piruvato; 3– Fluoroacetato de Sódio (SF,2 mM) inibe o ciclo do ácido tricarboxílico (AT); 4– Rotenona (ROT, 1M) e 5- Antimicina (ANT, 50nM), inibidores respectivamente dos complexos mitocondriais I e III. A expressão proteica do transportador GLUT2 foi avaliada em ilhotas pancreáticas. Os dados foram avaliados por análises de variância (ANOVA) ou Teste t de Student (p<0,05). Ratos MSG-SED desenvolveram obesidade, resistência à insulina e hipersecreção de insulina em relação aos ratos CON-SED. A natação evitou a hiperinsulinemia, corrigiu a resistência à insulina e atenuou o excesso de tecido adiposo em ratos MSG. Ilhotas pancreáticas de ratos MSG-SED apresentam hipertrofia, aumentada expressão do GLUT2, secretando cerca de 25% mais insulina em relação a ilhotas de ratos CON-SED. Em ambos os grupos a natação reduziu em média 25% a secreção de insulina estimulada por glicose. A natação atenuou a hipertrofia dos adipócitos, das ilhotas pancreáticas, bem como corrigiu a expressão do GLUT2 em ratos MSG-obesos. O efeito do bloqueador glicolítico foi mais acentuado em ilhotas de ratos MSG-SED, indicando maior ativação desta via. O bloqueio do complexo I mitocondrial teve efeito similar entre os grupos. Todavia a inibição do complexo III foi menos acentuada em ilhotas de ratos MSG-SED. Os bloqueadores do ciclo AT e transporte do piruvato não inibiram o controle secretor de insulina em ilhotas de todos os grupos; porém o α-CHC exerceu efeito paradoxal em ilhotas de ratos MSG-SED. Alterações da glicólise; do ciclo do AT e do complexo I mitocondrial parecem não estar envolvidas na menor resposta a glicose encontrada em ilhotas de ratos exercitados. Porém, aumento da participação do complexo III mitocondrial foi observado em ilhotas de ambos os grupos exercitados. O tratamento neonatal com MSG induz obesidade, dislipidemia e resistência à insulina, eventos atenuados e/ou revertidos pela natação. A hipersecreção de insulina é corrigida pelo exercício sem alterar a via glicolítica e/ou do ciclo do AT. Todavia, a natação parece elevar a participação do complexo III mitocondrial.

O diabetes mellitus tipo 1 é uma doença autoimune causada pela destruição das células β produtoras de insulina, presentes nas ilhotas pancreáticas, por células autorreativas do sistema imune. A opção de tratamento mais utilizada são injeções diárias de insulina exógena, o que configura um tratamento não curativo. Para alcançar a independência de insulina, alternativas como o transplante de ilhotas vêm sendo estudadas. Entretanto, a disponibilidade de pâncreas de doadores cadavéricos para o isolamento destas ilhotas é pequena e os métodos de isolamento, pouco eficazes, sendo necessários de 2 a 4 doadores para atingir o número adequado de ilhotas. Além disso, o transplante apresenta problemas relacionados à enxertia, devidos principalmente à baixa vascularização, o que leva à morte de células β nos primeiros dias pós-transplante. Desta forma, estudos explorando alternativas que aumentem a sobrevivência e a funcionalidade dos transplantes e diminuam o número de ilhotas exigido por receptor fazem-se muito necessários. As células-tronco mesenquimais apresentam propriedades interessantes para aplicação em terapia celular. Entre elas, destaca-se o efeito parácrino, que exerce diversas funções benéficas, como o aumento da vascularização, nos locais onde estas células estão presentes. Sendo assim, este trabalho explorou o co-transplante de ilhotas pancreáticas com células-tronco mesenquimais derivadas de tecido adiposo, para o tratamento do diabetes mellitus em modelo murino. Os resultados mostraram que a presença destas células no grupo que recebeu o co-transplante não aumentou a taxa de cura, em relação ao grupo que recebeu somente ilhotas. No entanto, o fenômeno de reversão do diabetes foi antecipado no grupo co-transplantado, o que sugere um possível efeito angiogênico das células-tronco adiposo-derivadas presentes neste grupo. Desta forma, conclui-se que estas células podem exercer atividades benéficas, quando co-transplantadas com ilhotas pancreáticas, para o tratamento do diabetes. / Type 1 diabetes mellitus is an autoimmune disease caused by destruction of insulin-producing β cells, present in pancreatic islets, by auto-reactive cells of the immune system. The most widely used treatment option are daily injections of insulin, which configures a non-curative treatment. To achieve insulin independence, alternatives such as islet transplantation have been studied. However, the availability of pancreas from cadaveric donors for the isolation of these islets is poor and the methods for isolation, ineffective, requiring 2 to 4 donors to achieve the appropriate number of islets. In addition, transplantation presents problems related to engraftment, mainly due to poor vascularization, which leads to β cell death in the first days after transplantation. Thus, studies exploring alternatives that increase the survival and function of transplants and reduce the number of islets required by the recipient are very necessary. Mesenchymal stem cells have interesting properties for application in cell therapy. Among them is the paracrine effect, which has several beneficial functions, such as promoting vascularization in the tissues where these cells are present. Thus, the present study explored the co-transplantation of pancreatic islets with mesenchymal stem cells derived from adipose tissue for the treatment of diabetes mellitus in mice. The results showed that the presence of these cells in the group that received co-transplantation did not increase the cure rate, compared to the group that received islets alone. However, the phenomenon of diabetes reversion was anticipated in co-transplanted animals, which suggests a possible angiogenic effect of adipose-derived stem cells present in this group. Thus, we conclude that these cells may exert beneficial functions when co-transplanted with pancreatic islets for the treatment of diabetes.

O tamanduá-bandeira Myrmecophaga tridactyla é uma espécie considerada &ldquo;vulnerável&rdquo; no Brasil, por estar ameaçado de extinção em algumas regiões do país. O presente projeto teve por objetivo identificar e caracterizar as estruturas macro e microscópicas do pâncreas nessa espécie. Para tanto, foram dissecados 16 pâncreas de tamanduás-bandeira provenientes do Hospital Veterinário &ldquo;Dr. Halim Atique&rdquo; do Centro Universitário de Rio Preto (UNIRP). As amostras coletadas, foram provenientes de casos de animais atendidos no referido Hospital e que vieram a óbito. O pâncreas situava-se no antímero esquerdo do corpo do animal, apresentava coloração pálida, corpo central e superfície lobulada. Acompanhava a curvatura ventricular maior do estomago aderindo-se na porção inicial do duodeno. Relaciona-se crâniodorsalmente com o baço e ventrículo gástrico, e caudoventralmente com a cápsula fibrosa renal (que aloja o rim esquerdo) e intestinos. Estruturalmente, o órgão demonstrou duas partes distintas: a primeira delas com características exócrinas, composta por ácinos pancreáticos e a segunda endócrina, formada pelas ilhotas pancreáticas encontradas nas regiões media, caudoventral e lobar esquerda. A analise ultraestrutural permitiu identificar nas células centro-acinosas do pâncreas vesículas com grânulos de zimogênio, mitocôndrias, Aparelho de Golgi e retículo endoplasmático rugoso / The giant anteater Myrmecophaga tridactyla is a species considered &#39;vulnerable&#39; in Brazil since it is threatened in some Brazilian regions. This study aimed to identify and characterize morphological structures of the pancreas in this species. For this, 16 anteaters pancreas from the Veterinary Hospital &quot;Dr. Halim Atique at University Center of Rio Preto (UNIRP), were dissected. All samples were from animals treated at the hospital which died of natural causes. The pancreas was located in the left antimere of the animal&rsquo;s body, being lobulated and having a pale color and central body. It followed the greater curvature of the stomach, adhering on the initial portion of the duodenum. It was craniodorsally related to the spleen and gizzard, and caudoventrally to the renal fibrous capsule (which houses the left kidney) and intestines. Structurally, the organ had two distinct parts: an exocrine, composed of pancreatic acini; and and endocrine, formed by pancreatic islets found in the medial, caudoventral and left lobar regions. The ultrastructural analysis allowed identifying the central-acinar pancreatic cells with vesicles zymogen granules, mitochondria, Golgi apparatus and rough endoplasmic reticulum

<p>Xenotransplantation, i.e. transplantation between different species, could be a possible solution to the present shortage of organ donors. The immunological response to a xenograft is strong and difficult to suppress. It is driven both by the humoral and cellular part of the immune system. The aim of this thesis was to characterise and modulate this response in a concordant mouse-to-rat model, using both vascularised and non-vascularised grafts.</p><p>Exposure of mouse cells or tissue to the circulation of a rat, either through transplantation or transfusions, easily evoked an immune response, consisting of IgM antibodies. A response that was aimed both at antigens present on mouse mononuclear cells and on erythrocytes. A non-immunosuppressed rat rejected a mouse heart graft within three days. The combined use of cyclosporine A (CyA) and deoxyspergualin (DSG) as immunosuppression prevented the rejection of vascularised heart transplants as well as of non-vascularised pancreatic islet grafts. This acceptance was sustained for the heart transplant also after the termination of DSG treatment, but not for the pancreatic islet graft. Furthermore, a second heart graft was accepted when transplanted under monotherapy with CyA 56-154 days after the first transplantation. This finding was interpreted as a humoral unresponsiveness, which could not be reproduced when the primary heart was substituted with a cellular graft, consisting of pancreatic islets or heart cells, or by blood transfusions. However, the rejection of a mouse heart after blood transfusions occurred in the absence of antibodies directed against mouse erythrocytes, in contrast to the observations in non-transfused animals. This indicates that a partial humoral tolerance restricted to the response against erythrocytes can be induced. This mechanism may offer a possibility to induce total humoral tolerance against a xenograft if the appropriate antigens are administered in conjunction with CyA and DSG.</p>

<p>Enterovirus (EV) infections have been associated with the pathogenesis of Type 1 Diabetes (T1D). However, the pathway(s) by which EV may induce or accelerate diabetes is not well understood. The purpose of this thesis was to obtain new information on the mechanism by which EV infections, with different strains of EV, could cause damage to the insulin-producing β-cells in isolated human islets and in a rat insulin-producing cell line (RINm5F). </p><p>Infection with EV strains isolated from T1D patients revealed replication/cell destruction in human islets and EV-like particles in the cytoplasm of the β-cell and infection with the isolates affected the release of insulin in response to glucose stimulation as early as three days post infection, before any decrease in cell viability was observed. A decrease in the induction/secretion of the chemokine RANTES in human islets during EV infection was also detected. When islets were cultured with nicotinamide (NA) the secretion of RANTES was increased irrespectively if the islets were infected or not. In addition, the degree of virus-induced cytolysis of human islets was reduced by NA, suggesting an antiviral effect of NA. Infection with EV strains revealed permissiveness to islet-derived cells. </p><p>All EV strains used for infection were able to replicate in the RIN cell clusters (RCC) but not in the RIN cells that were cultured as a monolayer. This might be due to the differences in expression of the Coxsackie-adenovirus receptor (CAR), which only could be detected on the RCC. Infection of RCC with a CBV-4 strain did not affect cell viability and did not induce nitric oxide (NO) production alone or with the addition of IFN-γ. This was in contrast to the results obtained with synthetic dsRNA, poly(IC), which induced NO, suggesting that synthetic dsRNA does not mimic enteroviral intermediate dsRNA.</p><p>During analyses performed with the samples from a family where the mother and one son where diagnosed with T1D on the same day, the results showed that the whole family had a proven EV infection at the time diagnosis.</p><p>To conclude, the ability of EV strains to replicate in RIN cells is dependent on the growth pattern of the cells and this may be due to the upregulation and/or changed expression pattern of CAR in these cells. In the RIN cells, contrary to artificial dsRNA, viral dsRNA does not induce NO. The isolated EV virus strains used were able to infect and affect human pancreatic islets in vitro. The chemokine RANTES is reduced during an EV infection of human pancreatic islets and NA causes upregulation of RANTES in infected and uninfected islets. </p>

This thesis focuses on enteroviral effects on human pancreatic islets. Most knowledge of viral effects on host cells relies on studies of immortalized cell lines or animal models. The islets represent a fundamentally different and less well studied cellular host. Also, enterovirus has been implicated in the etiology of type 1 diabetes (T1D). We show that when enterovirus replicates in human islets it activates innate immunity genes and induces secretion of the chemokines MCP-1 and IP-10. An important difference in activation of innate immunity by replicating EV and synthetic dsRNA is suggested, since the chemokine secretion induced by EV infection but not by dsRNA is reduced by female sex hormone. We also demonstrate a direct antiviral effect of nicotinamide, and even though this substance failed to prevent T1D in a large-scale study, this finding could have implications for the treatment/prevention of virus- and/or immune-mediated disease. We also had access to human pancreata from two organ donors with recent onset T1D and several donors with T1D-related autoantibodies, which gave us the opportunity to study ongoing pathogenic processes at and before the onset of T1D. Despite this, we could neither confirm nor reject the hypothesis that EV is involved in T1D development. Several observations, such as ultrastructural remodeling of the beta cell, activation of innate immunity, and immunopositivity to EV capsid protein 1, supported an ongoing virus infection, but direct evidence is still lacking. An interesting finding in the donors with recent onset T1D was that the islets were positively stained for insulin, but did not secrete insulin in response to glucose-stimulation. A similar effect was observed in EV-infected islets in vitro; EV destroyed islet function and insulin gene expression, but the islets still stained positive for insulin. This may be indicative of that a functional block in addition to beta cell destruction is involved in T1D pathogenesis. In conclusion, these studies of EV in isolated human islets in vitro support that this virus can cause T1D in vivo, but future studies will have to show if and how frequently this happens.