Diabetes mellitus (DM) ist eine Stoffwechselerkrankung, die durch eine chronische Hyperglykämie (erhöhter Blutzuckerspiegel) gekennzeichnet ist. Derzeit leiden mehr als 540 Millionen Menschen, die überwiegend in Ländern mit niedrigem und mittlerem Einkommen leben, an Diabetes, und es wird prognostiziert, dass die Inzidenzrate bis 2045 auf über 783 Millionen ansteigen wird, was auf eine große Krise im öffentlichen Gesundheitswesen hindeutet. Typ-1-Diabetes (T1D), eine chronische Autoimmunerkrankung, entsteht durch die von T-Zellen vermittelte Zerstörung der insulinproduzierenden β-Zellen in den Pankreasinseln, wodurch der Blutzuckerspiegel der Patienten nur unzureichend kontrolliert wird. T1D ist eine der häufigsten endokrinen und metabolischen Störungen im Kindesalter, und die Behandlungsmöglichkeiten umfassen die Verabreichung verschiedener Formen von exogenem Insulin durch unterschiedliche Modalitäten. Inseltransplantationen oder Transplantationen der gesamten Bauchspeicheldrüse sind nach wie vor die Standardtherapien, aber die Organknappheit stellt eine erhebliche Einschränkung dar. Als Alternative bieten sich humane pluripotente Stammzellen (hPSCs) für eine Diabetes- Zellersatztherapie an, aber die erzeugten inselähnlichen Cluster (SC-Inseln) sind funktionell noch nicht ausgereift. Zahlreiche Bemühungen, den Differenzierungsprozess zu verbessern, haben erhebliche Fortschritte gezeigt, aber die Herausforderungen in Bezug auf Effizienz, Funktionalität und Reifung bleiben bestehen. Ich stellte die Hypothese auf, dass (a) die Heterogenität von hPSC-abgeleiteten Pankreasvorläuferzellen (PPs) und (b) ihre Expansion sowie (c) die Förderung der Gefäßentwicklung in den SC-Inseln die β-Zellreifung und die geringe Umwandlungseffizienz von hPSCs in β-Zellen verbessern könnten. Um der Heterogenität der PP entgegenzuwirken, habe ich zunächst das Verfahren zur Differenzierung von hPSCs zu β-Zellen überarbeitet und dabei einen sehr hohen Anteil von 80-90% PDX1+/SOX9+/NKX6.1+ Zellen im PP-Stadium erreicht. Außerdem habe ich ein GMP-konformes, kostengünstiges und effizientes Expansionsverfahren verfeinert und vollständig etabliert, das eine konsistente exponentielle Expansion von PP-Zellen um das 2000-fache über 40 Tage ermöglicht. Bemerkenswerterweise kann dieses Verfahren die Anreicherung von expandierten PPs (ePPs) auf bis zu 80-90% PDX1+/SOX9+/NKX6.1+ Zellen vermitteln. Wichtig ist, dass ePPs mit ähnlicher Effizienz wie nicht-expandierte PP- Zellen in SC-Inseln mit funktionellen β-Zellen differenziert werden können. Ich stellte dann die Hypothese auf, dass die Einbeziehung sowohl von Endothelzellen (ECs) als auch von Perizyten (PCs) während der Differenzierung von PP-Zellen in einem Prozess, den ich als Tri-Kultur bezeichnete, die In-vivo-Entwicklung besser nachahmen und zu einer effizienten Differenzierung und Reifung von SC-Inseln führen würde. Um diese Hypothese der Vaskularisierung von SC-Inseln zu untersuchen, habe ich erfolgreich Verfahren zur gleichzeitigen Erzeugung von hochreinen (>95%) ECs und PCs etabliert, indem ich veröffentlichte Verfahren anpasste und modifizierte. So konnte ich sowohl PP-Zellen als auch vaskuläre Zellen aus derselben hPSC-Linie erzeugen. Im nächsten Schritt ermittelte ich das geeignete Zellverhältnis bei der gemeinsamen Ansammlung von PP-Zellen mit ECs und PCs sowie die geeignete Medienzusammensetzung, die das Überleben und die gemeinsame Entwicklung aller drei Zelltypen während S5-S7 ermöglichte. In den daraus resultierenden 'vaskularisierten' SC-Inseln (vSC-Inseln) waren die vaskulären Zellen gleichmäßig über die SC-Insel verteilt, was tatsächlich auf die Möglichkeit der Bildung von Mikrovaskulatur hindeutet. Die starke Expression endokriner Marker wurde beibehalten und die funktionelle Kompetenz der β-Zellen in den vSC-Inseln schien verbessert, was auf die potenziellen Vorteile der Vaskularisierung bei der Förderung der Inselfunktionalität hinweist. Die Entwicklung dieser vSC-Inseln ist ein wichtiger Schritt zur korrekten Modellierung der Inselentwicklung und eröffnet spannende Möglichkeiten zur Förderung der endokrinen Zellreifung in vitro sowie ihrer Integration nach der transplantation.:Summary ii Zusammenfassung iv Acknowledgements x List of Figures xii List of tables xiv List of abbreviations xv 1 INTRODUCTION 1 1.1 Classification and pathogenesis of diabetes 1 1.1.1 Type 1 Diabetes 2 1.1.2 Complications and challenges arising from diabetes 8 1.1.3 Approaches to address diabetes 9 1.1.4 Available therapeutic options and their constraints 12 1.2 Gastrulation 14 1.2.1 Patterning and germ layer specification in mammalian embryo 15 1.2.2 Patterning of the developing endoderm and gut tube formation 17 1.2.3 Emergence of alternative (hepatic and gut) lineages instead of pancreatic fate determination 19 1.2.4 Initiation of Pancreatic Fate and Morphogenesis 20 1.2.5 Primary transition 21 1.2.6 Secondary transition 23 1.2.7 Tertiary transition 27 1.2.8 Maturation of murine α and β cells during development 27 1.2.9 Differences in the developmental processes between humans and mice 28 1.2.10 Role of vasculature in islet development 30 1.3 Stem cells and their types: 33 1.3.1 Pluripotent stem cells: a promising source for β-cell replacement therapy 33 1.3.2 A long journey of hPSC towards generating β-cells 35 1.3.3 Significant differentiation protocols since last decade 37 1.3.4 Implementation of Stem cell therapies for T1D 41 1.3.5 Present status; clinical trials for the treatment of T1D, past and present 43 2 Material and methods 45 2.1 Cell culture 45 2.1.1 Coating of a matrix for the cell culture 45 2.1.2 Passaging of hESC cells 45 2.1.3 Freezing of and thawing of hPSCs 45 2.1.4 Freezing and thawing of PPs, ECs and PCs: 46 2.2 Differentiation of hPSCs to SC-islets using the Gavalas lab protocol 47 2.2.1 Seeding of hPSCs for PP differentiation 47 2.3 Expansion of PPs 49 2.4 Differentiation of hESCs into vascular cells 50 2.4.1 Differentiation of hESCs into ECS using ETV2 inducible line 50 2.4.2 Simultaneous differentiation of hESCs to ECs and PCs 50 2.5 RNA extraction, cDNA synthesis and RT-qPCR 51 2.5.1 RNA extraction 51 2.5.2 cDNA synthesis 52 2.5.3 RT-qPCR 52 2.6 Immunofluorescence analyses of monolayers cells, cryosections, and whole mounts 52 2.7 Flow cytometry (FC) of fixed (nuclear and surface markers) and live cells 54 2.8 Functionality assay for SC-islets 56 2.8.1 Static glucose-stimulated insulin secretion (GSIS) assay 56 2.8.2 C-peptide ELISA kit detection 56 2.9 Live Ca2+ imaging 57 2.10 Automated confocal imaging and analysis 57 2.11 Statistical analysis and graphs 57 3 Generation of SC-islets from hPSCs 58 3.1 hPSCs retained their pluripotency before the start of differentiation 58 3.2 Optimal seeding density is key to the successful differentiation 59 3.3 Efficient generation of definitive endoderm (DE) cells (S1) 60 3.4 Successful differentiation of DE cells into primitive gut tube (PGT) and posterior foregut (PF) 61 3.5 Generation of PDX1+/SOX9+/NKX6.1+ enriched PP population (S4) 64 3.6 Stagewise transcriptomic analysis of hPSCs to PP differentiation 66 3.7 PP cells differentiated into pancreatic endocrine progenitor (PEP) (S5) cells 68 3.8 Generation of SC-islets containing functional β cells 69 3.9 Inclusion of N21 in S7 increases INS expression, numbers of β-cells and β-cell responsiveness 71 4 Expansion of PPs under GMP-compatible condition 74 4.1 C6 expansion selects PDX1+/SOX9+/NKX6-1+ cells population across lines 74 4.2 GMP-compliant C6 shows similar growth kinetics in all three lines while maintaining chromosomal stability 77 4.3 The transcriptome of ePPs revealed their stabilization just after five passages 78 4.4 C6 promoted strong upregulation of GP2, a pancreatic progenitor marker, during expansion 79 4.5 Impact of expansion on progenitor transcription factors and ductal program activation 80 4.6 Expansion with C6 represses the endocrine program and alternative lineages 82 4.7 C6 ePPs differentiate into SC-islets containing functional β-cells 83 5 Generation of SC-islets containing endothelial cells and pericytes 85 5.1 The ETV2 inducible expression hPSC line 85 5.2 Efficient differentiation of hPSCs to ECs using the ETV2 inducible expression line 86 5.3 Co-culturing PPs with ECs till S7 89 5.4 Simultaneous generation of PCs and ECs from hPSCs 90 5.5 ECs and PCs can be expanded for three passages with similar efficiency in four different matrices 92 6 Discussion 101 7 Future outlook 110 8 Supplementary information 112 9 Bibliography 121 10 Anlage 1 144 11 Anlage 2 145

Diabetes mellitus tipo 1 resulta da produção insuficiente ou da ausência de insulina, decorrente da destruição de células β, por mecanismo auto-imune. O tratamento deste tipo de diabetes consiste na administração subcutânea de insulina exógena. Recentemente, foi demonstrado que o transplante de ilhotas pancreáticas é capaz de tornar o portador de diabetes tipo 1 independente de insulina exógena. Apesar do sucesso alcançado, a necessidade permanente de imunossupressão é uma das principais barreiras para que o transplante de ilhotas possa ser realizado em número maior de pacientes. Assim, o desenvolvimento de novas metodologias que evitem a rejeição do enxerto, como o macro e o microencapsulamento de ilhotas, continua sendo crucial para o estabelecimento definitivo do transplante de ilhotas como opção terapêutica no tratamento de diabetes tipo 1. Neste trabalho, foi padronizado um modelo animal para avaliar, in vivo, a funcionalidade das ilhotas pancreáticas humanas isoladas e purificadas na Unidade de Ilhotas Pancreáticas Humanas do IQUSP. Ratos NIH nude foram tornados diabéticos através de injeção de estreptozotocina para o implante de ilhotas pancreáticas humanas nuas e microencapsuladas. As ilhotas foram microencapsuladas em Biodritina, um novo heteropolissacarídeo patenteado e cedido ao nosso laboratório, tendo sido possível padronizar a produção de microcápsulas uniformes e homogêneas, com tamanho médio entre 400µm e 600 µm. A reversão do diabetes ocorreu em 24% dos ratos nude transplantados com ilhotas pancreáticas humanas nuas. Por outro lado, não observamos reversão do diabetes quando ilhotas encapsuladas foram implantadas, apesar do teste de atividade funcional realizado in vitro ter demonstrado que elas continuam a secretar insulina e a responder ao estímulo com glicose após o encapsulamento. Para elucidar este efeito, cápsulas vazias foram implantadas em ratos nude e em ratos imunocompetentes, os quais desenvolveram processo inflamatório acompanhado de processo fibrótico no local do implante. Estudo imuno-histoquímico está sendo realizado para esclarecer a natureza e a intensidade destes processos. / Type 1 diabetes mellitus results from insufficient or absence of insulin production, as a consequence of destruction of pancreatic β cells, by an auto-imune mechanism. Treatment for this type of diabetes consists of subcutaneous administration of exogenous insulin. Recently, it has been demonstrated that pancreatic islet cell transplantation is capable of rendering type I diabetic patients independent of exogenous insulin. However, in spite of the success achieved, permanent immunosuppression is still required, being the main barrier to expand this treatment to a large number of patients. Therefore, development of new technologies, such as islet macro and microencapsulation to avoid rejection of the tissue implanted, is still crucial for definitive establishment of islet transplantation as a therapeutic alternative for type I diabetes. In the present work, an animal model was established for in vivo evaluation of the functional ability of human pancreatic islets, which were isolated and purified at the Human Pancreatic Islet Unit of the University of São Paulo Chemistry Institute. Diabetes was induced in NIH nude rats through streptozotocin injection followed by implantation of naked or microencapsulated human pancreatic islets. Biodritin, a new and patented heteropolyssaccaride was used to microencapsulate the islets. The production of uniform and homogeneous microcapsules with diameters in the range of 400µm e 600 µm was successfully established. Reversion of diabetes occurred in 24% of the nude rats transplanted with human pancreatic islets. On the other hand, no reversion of diabetes was observed when encapsulated islets were implanted, although their functional activity in vitro indicated that they secreted insulin and responded to glucose stimulation upon encapsulation. In order to elucidate this effect, empty capsules were implanted in nude rat and in immunocompetent rats, both of which developed an inflammatory process accompanied by a fibrotic process in the site of the implant. Immunohistochemical studies are underway to address the nature and the intensity of these inflammatory processes.

Diabetes mellitus tipo 1 resulta da produção insuficiente ou da ausência de insulina, decorrente da destruição de células β, por mecanismo auto-imune. O tratamento deste tipo de diabetes consiste na administração subcutânea de insulina exógena. Recentemente, foi demonstrado que o transplante de ilhotas pancreáticas é capaz de tornar o portador de diabetes tipo 1 independente de insulina exógena. Apesar do sucesso alcançado, a necessidade permanente de imunossupressão é uma das principais barreiras para que o transplante de ilhotas possa ser realizado em número maior de pacientes. Assim, o desenvolvimento de novas metodologias que evitem a rejeição do enxerto, como o macro e o microencapsulamento de ilhotas, continua sendo crucial para o estabelecimento definitivo do transplante de ilhotas como opção terapêutica no tratamento de diabetes tipo 1. Neste trabalho, foi padronizado um modelo animal para avaliar, in vivo, a funcionalidade das ilhotas pancreáticas humanas isoladas e purificadas na Unidade de Ilhotas Pancreáticas Humanas do IQUSP. Ratos NIH nude foram tornados diabéticos através de injeção de estreptozotocina para o implante de ilhotas pancreáticas humanas nuas e microencapsuladas. As ilhotas foram microencapsuladas em Biodritina, um novo heteropolissacarídeo patenteado e cedido ao nosso laboratório, tendo sido possível padronizar a produção de microcápsulas uniformes e homogêneas, com tamanho médio entre 400µm e 600 µm. A reversão do diabetes ocorreu em 24% dos ratos nude transplantados com ilhotas pancreáticas humanas nuas. Por outro lado, não observamos reversão do diabetes quando ilhotas encapsuladas foram implantadas, apesar do teste de atividade funcional realizado in vitro ter demonstrado que elas continuam a secretar insulina e a responder ao estímulo com glicose após o encapsulamento. Para elucidar este efeito, cápsulas vazias foram implantadas em ratos nude e em ratos imunocompetentes, os quais desenvolveram processo inflamatório acompanhado de processo fibrótico no local do implante. Estudo imuno-histoquímico está sendo realizado para esclarecer a natureza e a intensidade destes processos. / Type 1 diabetes mellitus results from insufficient or absence of insulin production, as a consequence of destruction of pancreatic β cells, by an auto-imune mechanism. Treatment for this type of diabetes consists of subcutaneous administration of exogenous insulin. Recently, it has been demonstrated that pancreatic islet cell transplantation is capable of rendering type I diabetic patients independent of exogenous insulin. However, in spite of the success achieved, permanent immunosuppression is still required, being the main barrier to expand this treatment to a large number of patients. Therefore, development of new technologies, such as islet macro and microencapsulation to avoid rejection of the tissue implanted, is still crucial for definitive establishment of islet transplantation as a therapeutic alternative for type I diabetes. In the present work, an animal model was established for in vivo evaluation of the functional ability of human pancreatic islets, which were isolated and purified at the Human Pancreatic Islet Unit of the University of São Paulo Chemistry Institute. Diabetes was induced in NIH nude rats through streptozotocin injection followed by implantation of naked or microencapsulated human pancreatic islets. Biodritin, a new and patented heteropolyssaccaride was used to microencapsulate the islets. The production of uniform and homogeneous microcapsules with diameters in the range of 400µm e 600 µm was successfully established. Reversion of diabetes occurred in 24% of the nude rats transplanted with human pancreatic islets. On the other hand, no reversion of diabetes was observed when encapsulated islets were implanted, although their functional activity in vitro indicated that they secreted insulin and responded to glucose stimulation upon encapsulation. In order to elucidate this effect, empty capsules were implanted in nude rat and in immunocompetent rats, both of which developed an inflammatory process accompanied by a fibrotic process in the site of the implant. Immunohistochemical studies are underway to address the nature and the intensity of these inflammatory processes.

In addition to host defense, alternative functions of immune cells are emerging. Immune cells are crucial during healing of injured tissue, in formation of new blood vessels, angiogenesis, and also in maintaining the balance in inflammation having immune regulating functions. Over the last decade a higher degree of heterogeneity and plasticity of immune cells have been reported and immune cells develop different characteristics in different situations in vivo. This thesis investigates roles for immune cells in situations of muscle hypoxia and reduced blood perfusion, wound healing in skin and at sites of transplantation of allogeneic islets of Langerhans and on top of this, ways to steer immune cell function for future therapeutic purposes. A specific neutrophil subset (CD49d+VEGFR1+CXCR4high) was found to be recruited to VEGF-A released at hypoxia and these neutrophils were crucial for functional angiogenesis. In muscle with restricted blood flow macrophages were detected in perivascular positions and started to express aSMA and PDGFR1b and were found to directly assist in blood flow regulation by iNOS-dependent NO production. This essential function in muscle regain of function could be boosted by plasmid overexpression of CXCL12 where the effect of these macrophages chaperoning the vasculature was amplified improving limb blood flow regulation. The effect on macrophages accelerating tissue regeneration being amplified by CXCL12 was tested in a model of cutaneous wound healing where the administration of CXCL12 was optimized for high bioavailability. In the skin, CXCL12-treatment induced accumulation of TGFb-expressing macrophages close to the wound driving the healing process, and subsequently the wounds healed with an efficiency never reported before. In the last study means to circumvent systemic immune suppressive therapy required in allogeneic transplantation was investigated. Allogeneic islets of Langerhans transplanted to muscle were immediately destroyed by the host immune system. Co-transplanting islets and CCL22-encoding plasmids we could curb this fast rejection for 10 days by accumulating CD4+CD25+FoxP3+ regulatory T lymphocytes at the site for transplantation preventing islet grafts from being attacked by the host cytotoxic T lymphocytes. In summary this thesis outlines distinct immune cell subsets being essential for regain of tissue function in hypoxia, ischemia and post injury and ways to amplify specific immune cell functions in these situations that are feasible for clinical use.

A privação dos hormônios sexuais, natural ou induzida, contribui para o aparecimento de diversas desordens metabólicas e endócrinas. Esse estudo investigou se a suplementação em dose única com DHEA, esteróide mais abundante em humanos, melhora a sensibilidade à insulina, bem como sua secreção e ou tolerância à glicose em ratas castradas alimentadas com dieta hiperlipídica (OHL). A castração induziu a perda da proteção fisiológica das fêmeas contra o ganho de peso. O tratamento com DHEA não promoveu alterações sobre esse parâmetro, porém, corrigiu a elevação na concentração de insulina plasmática e o índice HOMA IR, além da constante de decaimento de glicose, kitt. Os animais castrados apresentaram aumento da área da ilhota. DHEA não alterou essa condição. No entanto, as ilhotas das ratas tratadas com DHEA apresentaram aumento do grau de fosforilação da proteína Akt e melhora da capacidade secretória estática de insulina. Esse estudo sugere o uso do DHEA como alternativa protetora sobre a sensibilidade a insulina em fêmeas desprovidas de ovários. / Natural and induced privation of sexual hormones contributes to the development of several metabolic and endocrine disorders. The present study evaluated if DHEA supplementation, the most abundant steroid in humans, would improve the insulin sensitivity and secretion as well as the glucose tolerance, in high fat diet fed ovariectomized rats (OHL). Ovariectomy (OVX) reduced the physiological female protection against the weight gain. Although no effect upon adipose depot-specific action of DHEA has been found, DHEA has corrected the blood insulin levels and HOMA IR. In addition, DHEA has improved peripheral insulin action by the glucose disappearance rate, kitt. The islets area was increased in all ovariectomized groups. Pancreatic islets from DHEA-treated rats showed an increased in the Akt serine phosphorylation status and restored glucose-stimulated insulin secretion. Our results suggest that DHEA can promote protective effects by increasing the insulin sensitivity in females castrated rats exposed to health risk factors.

As ilhotas pancreáticas têm em sua constituição as células <font face=\"Symbol\">b pancreáticas, as quais têm como função secretar insulina. A melatonina é secretada pela glândula pineal. No entanto, a ausência da melatonina, por meio da pinealectomia (PINX), induz diversas alterações nas funções celulares. A NAD(P)H oxidase é responsável pela produção de ânions superóxido. Nosso estudo teve como objetivo avaliar uma possível modulação da NAD(P)H oxidase pela PINX no Zeitgeber Time 6 e 18. Os resultados demonstram que a PINX induz alterações na funcionalidade das ilhotas pancreáticas, alterando a secreção de insulina estimulada pela glicose, o metabolismo da glicose, e o conteúdo de espécies reativas de oxigênio (EROs). Entretanto, não induz alterações nos respectivos ZTs na expressão protéica das subunidades p22phox, p47phox e gp91phox. Demonstramos que a pinealectomia induz alteração no padrão rítmico metabólico e secretório nas ilhotas isoladas, tendo a NAD(P)H oxidase uma possível participação no desenvolvimento nas alterações observadas. / Pancreatic islets are constituted by pancreatic cells, which main function is to secrete insulin. Melatonin is secreted by the pineal gland. However, the absence of melatonin by pinealectomy (PINX) induces several changes in cellular functions. The NAD(P)H oxidase is responsible for producing superoxide. Our study aimed to evaluate the possible modulation of NAD(P)H oxidase by pinealectomy in Zeitgeber Time 6:18. Our results show that the absence of melatonin induced by pinealectomy induces changes in the functionality of pancreatic islets, such as the insulin secretion stimulated by glucose, the glucose metabolism, and the content of reactive oxygen species (ROS). However, it does not induce changes in their respective Zts in the protein expression of the subunits p22phox, p47phox and gp91phox. We demonstrated that pinealectomy induces changes in metabolic and secretory rhythm pattern in isolated islets, being the NAD(P)H oxidase a possibly responsible for the changes observed.

Durante a gravidez, o pâncreas endócrino materno sofre alterações morfológicas e funcionais que resultam no aumento da massa de células beta e da secreção de insulina. Nos estágios finais da gestação ocorre aumento dos níveis plasmáticos de glicocorticóides que resulta na diminuição da secreção e da proliferação das células beta. Este fenômeno, que ocorre no período compreendido entre o final da gravidez e o inicio da lactação, promove a reversão fisiológica da adaptação funcional que se fez necessária durante a gravidez. Assim, estudamos mecanismos moleculares envolvidos na redução de proliferação destas células. As proteínas cinases reguladas por sinais extracelulares (ERK) estão envolvidas no crescimento e sobrevida celular. Os resultados mostram que o glicocorticóide sintético, dexametasona, diminui a proliferação de células beta e, para isto, induz diminuição da fosforilação das ERK-1/2 por meio do aumento da expressão de uma fosfatase de MAPK (MKP-1). Este mecanismo deve estar envolvido no remodelamento pancreático pós-natal induzido pelos glicocorticóides. / During pregnancy, maternal pancreatic islets undergo morphofunctional changes that increase beta cell mass and insulin secretion. At late stages of pregnancy there is an increase in plasma glucocorticoid levels that inhibit beta cell proliferation and beta cell function. This situation, which occurs in a period between late pregnancy and early stages of lactation, counteracts the functional gain established throughout pregnancy. In this work we studied the molecular mechanisms involved in the impaired beta cell proliferation. The extracellular regulated kinases (ERKs) are involved in cellular growth and survival. Our results show that dexametasone, a synthetic glucocorticoid, inhibits proliferation by a mechanism that includes up regulation of a dual specificity phosphatase (MKP1). This, by extension, impairs ERK1/2 activation. This mechanism could take part in the induced-glucocorticoid reestablishment of endocrine pancreatic mass after parturition.

O efeito de alterações no estado de óxido-redução (redox), tanto pelo aumento no estado oxidativo quanto pelo aumento no estado redutor, foi avaliado sobre a funcionalidade de ilhotas pancreáticas, através da análise da secreção de insulina estimulada pela glicose (GSIS), metabolismo da glicose e oscilações intracelulares de cálcio. O aumento no estado oxidativo inibiu a funcionalidade da célula pancreática. Entretanto, diminuição no estado oxidativo pela adição de antioxidantes exerceu efeito dual sobre a funcionalidade da célula <font face=\"Symbol\">&#946 pancreática, na qual pequenas alterações no estado redox estimularam a GSIS, enquanto alterações maiores suprimiram este efeito positivo. Adicionalmente, o conteúdo das espécies reativas de oxigênio (EROs) foi modulado por mudanças na concentração de glicose. Agudamente, o aumento na concentração de glicose suprimiu o conteúdo de EROs, que pôde ser correlacionada com o aumento na atividade da via de formação de NADPH, a via das pentoses-fosfato. Sob estes aspectos, alterações no estado redox podem ser parte do processo da GSIS. / The effect of changes in the oxidation/reduction (redox) state over pancreatic islet function was analyzed by shifts toward oxidative or reducing environments. Pancreatic cell function was analyzed by glucose-stimulated insulin secretion (GSIS), glucose metabolism and intracellular calcium oscillations. Redox modulation favoring the oxidative state inhibited pancreatic cell function. However, the suppression of the oxidative state by antioxidant treatment exerted a dual effect on pancreatic <font face=\"Symbol\">&#946 cell function, where small changes were positively correlated with an increase in insulin secretion, while higher changes suppressed GSIS. Additionally, the reactive oxygen species (ROS) content was modulated by changes in glucose concentration. Increasing concentrations of glucose acutely suppressed ROS content, what was correlated with the activation of the NADPH source, the pentose-phosphate pathway. Thus, the intracellular adjustment of ROS content may be part of the insulin secretion mechanism in response to glucose.

O transplante (Tx) de ilhotas pancreáticas (IP) é uma atraente alternativa para o tratamento do diabetes melito tipo 1. No entanto, para evitar a rejeição há necessidade de imunossupressão. Uma nova idéia de tolerância surge a partir do paradoxo imunológico, onde a mãe, imunologicamente competente, não rejeita o embrião durante a gravidez. Uma das hipóteses é que células da placenta expressam a molécula IDO, a qual protege o embrião do ataque imunológico materno. O objetivo do estudo foi analisar o efeito da indução da expressão da IDO em IP em transplante experimental de IP. Para tanto, as seguintes etapas de padronização foram necessárias. Etapa 1: Padronização da perfusão e digestão do tecido pancreático de rato e determinação do método para a purificação das IP, comparando-se diferentes gradientes de densidade: descontínuo de Ficoll, contínuo de Ficoll e contínuo de iodixanol. Foi demonstrado que o gradiente contínuo de iodixanol fornece maior pureza e maior número de IP íntegras e funcionais. Etapa 2: Padronização do Tx experimental de IP sob a cápsula renal para avaliação do número mínimo de IP transplantadas para reverter o diabetes induzido por estreptozotocina, definido como glicemia >300mg/Kg. Foram transplantadas entre 200 a 3.000 IP por experimento. A rejeição das IP foi analisada pela sobrevida das IP (permanência da glicemia <300mg/dL), tanto em Tx isogênico (Lewis-Lewis) como em alogênico (Sprague-Dawley-Lewis). Para reverter o diabetes foram necessárias no mínimo 2.500 IP. No transplante entre ratos isogênicos (n=6) não houve rejeição das IP. Já no transplante entre animais alogênicos (n=12), as IP apresentaram uma curta sobrevida pós-Tx (11±1 dias; p<0,01 vs. Tx isogênico). Dez dias pós-Tx, houve um grande infiltrado de macrófagos e linfócitos T no enxerto alogênico e uma diminuição significativa da expressão de insulina (p<0,001 vs. Tx isogênico). Etapa 3: Construção do vetor de expressão para IDO. A partir de RNA extraído de placenta de rata no 10º dia de gestação, foi amplificada a seqüência completa do cDNA para IDO, utilizando-se RT-PCR. Em seguida, o cDNA para IDO foi inserido em vetor de expressão (vetor-IDO). Etapa 4: Transfecção do vetor-IDO nas IP. O vetor-IDO foi introduzido nas IP através de lipofecção (Lipofectamina 2000), testando-se diferentes concentrações do vetor-IDO (0, 0,5, 1 e 10 ng/uL) e diferentes períodos de incubação (1h, 15h e 24h). A expressão de IDO nas IP foi confirmada por RT-PCR e imuno-histoquímica. A incubação com 10 ng/uL de vetor-IDO durante 24h foi eficaz para induzir a expressão de IDO nas IP, confirmada a nível de RNAm (RT-PCR) e de proteína (imuno-histoquímica). A eficiência da transfecção em nível funcional foi confirmada pela degradação de triptofano em cultura (dosagem de triptofano por HPLC). Etapa 5: Onze transplantes alogênicos (Sprague-Dawley-Lewis) com IP transfectadas com vetor-IDO foram realizados para analisar o efeito da IDO. Três animais foram sacrificados para análise de imuno-histoquímica e 8 animais foram acompanhados por 45 dias. A sobrevida das IP transfectadas com vetor-IDO foi significativamente maior comparada com a sobrevida de IP não-transfectadas (p<0,01). O estudo conclui que a expressão da IDO protege as IP aumentando a sobrevida das IP. / Transplantation (Tx) of pancreatic islets (PI) is an attractive alternative of treatment for type 1 diabetes mellitus. However, continuous immunossupression is necessary in order to avoid allograft rejection. A new idea of tolerance is based on the immunological paradox, during pregnancy, in that the mother, immunologically competent, does not reject the semi-allogeneic fetus. The hypothesis is that the placenta produces IDO molecules, which protect the embryos against the maternal immunologic attack. The aim of this study was to analyze the effect of the induction of the IDO expression into PI in an experimental model of PI transplantation. The following steps for standardization were necessary. Step 1: Besides the standardization of the rat pancreas perfusion and digestion, the best method for purification of the PI was determined, comparing several density gradients: Ficoll discontinuous, Ficoll continuous and iodixanol continuous. The iodixanol continuous gradient was able to provide high purity and a high number of intact and functional PI. Step 2: The transplantation of the PI between rats was established determining the minimal number of PI to reverse the diabetes (glycemia > 300mg/dL) induced by streptozotocin. In addition, the rejection was analyzed by PI survival (time with glycemia <300mg/dL) in syngeneic (Lewis-Lewis) and allogeneic (Sprague-Dawley-Lewis) transplantation. To reverse the diabetes at least 2,500 PI were necessary. Transplantation between syngenic rats (n=6) disclosed no rejection of the PI. In the allogeneic transplantation (n=12), the PI had a short survival (11±1 days). Ten days post-Tx, a higher number of macrophages and T lymphocytes were observed in the grafts, accompanied by very low insulin expression. Step 3: The expression vector for IDO was constructed from RNA extracted from rat placenta. RT-PCR was carried out to amplify the IDO cDNA, which was inserted into expression vector (IDO vector). Step 4: The IDO vector was introduced into PI through lipofection (Lipofectamine 2000) analyzing several concentrations of the IDO vector (0, 0.5, 1.0 and 10 ng/uL) and several periods of incubation (1h, 15h e 24h). The IDO expression in PI was confirmed by RT-PCR and immunohistochemistry. The incubation with 10 ng/uL of IDO vector during 24h was efficient to induce IDO expression in PI. The function of the IDO was confirmed by tryptofan degradation in culture (measurement of tryptofan by HPLC). Step 5: Eleven allogenic transplants (Sprague-Dawley to Lewis) of PI expressing IDO were performed to analyze the effect of the IDO in the rejection. Eight animals were accompanied for 45 days, whereas three were sacrificed after 10 days for immunohistochemistry analysis. Finally, the survival of the PI expressing IDO was significantly higher than nontransfected PI. The study concludes that the induction of the IDO into PI protects the PI increasing the PI survival.

Na gestação há aumento da proliferação e redução da apoptose das células b pancreáticas. Prolactina (PRL) desencadeia estas mudanças, que são revertidas após o parto mesmo na presença de PRL. In vitro, dexametasona (DEX) se contrapõe a PRL. Avaliamos se o estresse do retículo endoplasmático (ERE) está envolvido na apoptose do pós-parto e se os glicocorticóides (GC) participam deste mecanismo. A fragmentação do DNA aumenta no 3° dia pós-parto (L3), em paralelo com a diminuição de pAKT e aumento do TRB3, indutor da apoptose por ERE. BiP, ATF4, CHOP, e a ligação de CHOP e CHOP-ATF4 no promotor do TRB3 aumentam em L3. O inibidor do ERE PBA restaurou os níveis de pAKT e CHOP e inibiu a apoptose. Células RINm5F tratadas com DEX (24h) têm aumento de BiP e ATF4, de p-eIF2 e do XBP-1 ativo. DEX também induz TRB3, mas inibe a ligação de CHOP ao TRB3. O tratamento por 72h não altera p-eIF2a, diminui XBP-1 ativo e promove apoptose, único evento revertido pela PRL. Concluímos que a apoptose das ilhotas em L3 é desencadeada por ERE, mas os GC não induzem este mecanismo. / During gestation occurs increase on the proliferation and apoptosis reduction of pancreatic b cells. Prolactin (PRL) promotes these changes which are reverted after delivery. Dexametasone (DEX) in vitro opposed to PRL. We evaluate whether endoplasmatic reticulum stress (ERS) was involved on post-delivery apoptosis and glycocorticoids (GC) participate on this mechanism. DNA fragmentation increased on the 3rd day post-delivery (L3), in parallel with pAKT diminution and inductor of apoptosis-TRB3 augment by ERS. BiP, ATF4, CHOP along with binding of CHOP and CHOP-ATF4 to the TRB3 promoter increased in L3. ERS inhibitor-PBA restored pAKT, CHOP levels and inhibited apoptosis. RINm5F cells with DEX (24h) showed increase in BiP, ATF4, p-eIF2 and in active XBP-1. DEX induced TRB3, but inhibited the binding of CHOP to TRB3. The 72h treatment did not alter p-eIF2a, diminished active XBP-1 and promoted apoptosis; the unique event reverted by PRL. We concluded that apoptosis of islets in L3 is generated by ERS; nevertheless this mechanism is not induced by GC.