Le récepteur DcR3 (Decoy receptor 3) est un membre de la famille des récepteurs aux facteurs de nécrose tumorale (TNF). Il est fortement exprimé dans les tissus humains normaux ainsi que les tumeurs malignes. DcR3 est un récepteur pour trois ligands de la famille du TNF tels que FasL, LIGHT et TL1A. Étant une protéine soluble donc dépourvue de la portion transmembranaire et intracytoplasmique, le récepteur DcR3 est incapable d’effectuer une transduction de signal intracellulaire à la suite de son interaction avec ses ligands. De ce fait, DcR3 joue un rôle de compétiteur pour ces derniers, afin d’inhiber la signalisation via leurs récepteurs fonctionnels tels que Fas, HVEM/LTbetaR et DR3. Lors de nos précédentes études, nous avons pu démontrer, que DcR3 pouvaist moduler la fonction des cellules immunitaires, et aussi protéger la viabilité des îlots de Langerhans. À la suite de ces résultats, nous avons généré des souris DcR3 transgéniques (Tg) en utilisant le promoteur du gène β-actine humaine afin d’étudier plus amplement la fonction de ce récepteur. Les souris Tg DcR3 ont finalement développé le syndrome lupus-like (SLE) seulement après l’âge de 6 mois. Ces souris présentent une variété d'auto-anticorps comprenant des anticorps anti-noyaux et anti-ADN. Elles ont également manifesté des lésions rénales, cutanées, hépatiques et hématopoïétiques. Contrairement aux modèles de lupus murin lpr et gld, les souris DcR3 sont plus proche du SLE humain en terme de réponse immunitaire de type Th2 et de production d'anticorps d'anti-Sm. En péus, nous avons constaté que les cellules hématopoïétiques produisant DcR3 sont suffisantes pour causer ces pathologies. DcR3 peut agir en perturbant l’homéostasie des cellules T pour interférer avec la tolérance périphérique, et ainsi induire l'autoimmunité. Chez l'humain, nous avons détecté dans le sérum de patients SLE des niveaux élevés de la protéine DcR3. Chez certains patients, comme chez la souris, ces niveaux sont liés directement aux titres élevés d’IgE. Par conséquent, DcR3 peut représenter un facteur pathogénique important du SLE humain. L’étude des souris Tg DcR3, nous a permis aussi d’élucider le mécanisme de protection des îlots de Langerhans. Le blocage de la signalisation des ligands LIGHT et TL1A par DcR3 est impliqué dans une telle protection. D'ailleurs, nous avons identifié par ARN microarray quelques molécules en aval de cette interaction, qui peuvent jouer un rôle dans le mécanisme d’action. Nous avons par la suite confirmé que Adcyap1 et Bank1 joue un rôle critique dans la protection des îlots de Langerhans médiée par DcR3. Notre étude a ainsi élucidé le lien qui existe entre la signalisation apoptotique médiée par Fas/FasL et la pathogénèse du SLE humain. Donc, malgré l’absence de mutations génétiques sur Fas et FasL dans le cas de cette pathologie, DcR3 est capable de beoquer cette signalisation et provoquer le SLE chez l’humain. Ainsi, DcR3 peut simultanément interférer avec la signalisation des ligands LIGHT et TL1A et causer un phénotype plus complexe que les phénotypes résultant de la mutation de Fas ou de FasL chez certains patients. DcR3 peut également être utilisé comme paramètre diagnostique potentiel pour le SLE. Les découvertes du mécanisme de protection des îlots de Langerhans par DcR3 ouvrent la porte vers de nouveaux horizons afin d'explorer de nouvelles cibles thérapeutiques pour protéger la greffe d'îlots. / Decoy receptor 3 (DcR3) is a member of the tumor necrosis factor (TNF) receptor family, and is widely expressed in human normal tissues and malignant tumors. It is a decoy receptor of three TNF family members, i.e., FasL, LIGHT and TL1A. The interaction of DcR3 and its ligands will not transmit signal into cells via DcR3 because DcR3 is a soluble protein without a transmembrane and intracellular segment. Thereby, DcR3 competitively inhibits signaling through three functional receptors, i.e., Fas, HVEM/LTbetaR and DR3. In previous studies, we found that DcR3 could modulate immune cell function, and protect islet viability. Herein, we generated DcR3 transgenic (Tg) mice driven by the human β-actin promoter to further investigate the function of DcR3. Interestingly, the DcR3 Tg mice developed a lupus-like syndrome at 6 months of age. They presented a variety of autoantibodies including anti-nucleus and anti-dsDNA antibodies. They also manifested renal, dermal, hepatic and hematopoietic lesions. Compared to lpr and gld mouse lupus models, DcR3 Tg mice more closely resembled human SLE in terms of Th2-biased immune response and anti-Sm antibody production. Furthermore, we found that DcR3-producing hematopoietic cell were sufficient to cause these pathological changes. Mechanistically, DcR3 may break T-cell homeostasis to interfere with peripheral tolerance, and then induce autoimmunity. In humans, we detected high DcR3 levels in SLE patient sera. The high DcR3 levels were related to elevated IgE titer in some SLE patients, as was the case in the mouse model. Therefore, DcR3 may represent an important pathogenetic factor of human SLE. Utilizing the DcR3 Tg mouse, we further elucidated the mechanism by which DcR3 protected islets from primary nonfunction (PNF). Blocking of LIGHT and TL1A signaling by DcR3 are involved in such protection. Moreover, by mRNA microarray we identified possible downstream molecules, which may mediate such protection. We confirmed that Adcyap1 and Bank1 played critical roles in mediating DcR3’s effect in islet protection. Our studies resolved a puzzle about the relationship between the Fas/FasL apoptosis signaling pathway and the pathogenesis of human SLE. DcR3 can block Fas/FasL pathway even if there is no genetic mutation in Fas and FasL. DcR3 can simultaneously interfere with LIGHT and TL1A signaling to cause a more complex phenotype than the simple Fas or FasL mutation in patients. DcR3 can also be employed as a potential diagnostic parameter for SLE. The discovery of the mechanism of DcR3 in protecting islets allows us to explore novel therapeutic targets to protect islet graft.

O transplante de ilhotas pancreáticas humanas é considerado uma estratégia promissora para curar pacientes portadores de Diabetes Mellitus tipo 1. Entretanto, sua eficiência é dramaticamente afetada pelo rendimento das ilhotas no processo de isolamento/purificação e pela viabilidade das células após o transplante. As ilhotas pancreáticas isoladas são obtidas através da perfusão do pâncreas com colagenase e purificação em gradiente de densidade. As espécies reativas de oxigênio (ERO) exercem um papel importante durante a obtenção e o transplante de ilhotas pancreáticas humanas, contribuindo significativamente para diminuir a viabilidade dessas células. Nesse trabalho foram avaliadas as respostas oxidativas de ilhotas pancreáticas humanas durante os processos de isolamento/purificação e cultivo. As atividades da superóxido dismutase (SOD), da catalase, bem como os níveis de oxidação em proteínas mostraram-se, na maioria dos casos, aumentados, principalmente durante a etapa de purificação das ilhotas em gradiente de Ficoll e no período de cultura das ilhotas. Esses resultados indicam que a purificação em gradiente de Ficoll parece ser uma etapa crítica de geração das ERO, assim como longos períodos de cultivo. Porém, verificou-se que influências advindas dos diferentes doadores (idade, causa- mortis, estilo de vida, etc.) e condições de preservação do órgão (tempo de isquemia, solução de conservação, etc.) poderiam estar relacionadas à discrepância de alguns resultados encontrados. Com o intuito de minimizar tais variáveis, optou-se por estudar os efeitos relacionados ao Ficoll em células de insulinoma INS-1E, um modelo celular fisiologicamente semelhante. Para tanto, as atividades das enzimas antioxidantes SOD, catalase, glutationa peroxidase (GPx) e glutationa redutase (GR), assim como os danos oxidativos em proteínas e lipídeos, os níveis de glutationa reduzida (GSH) e de glutationa oxidada (GSSG), a viabilidade celular e os níveis de algumas enzimas envolvidas no processo apoptótico como p38, JNK-1, ERK 1-2 e PI3-K expostas a polissacarose (1100 mg/mL), um genérico do Ficoll, foram determinadas. De acordo com os resultados, as atividades da SOD, catalase e GPx presentes em amostras expostas a polissacarose mostraram-se aumentadas. Em cultura, a atividade de isoforma mitocondrial da SOD (Mn-SOD) de células INS-1E correspondeu a 50% da atividade total da SOD. Na presença da polissacarose, a atividade da Mn-SOD aumentou para 80% do total. Além disso, a oxidação de lipídios e de proteínas aumentou e os níveis de GSH e GR diminuíram discretamente. Estes resultados mostraram que a exposição dessas células a polissacarose está associada com o estresse oxidativo. Entretanto, tal exposição não foi responsável pela diminuição da viabilidade celular embora os níveis protéicos de JNK-1, ERK1-2 e PI3-K tenham se mostrado consideravelmente aumentados e os níveis de p38, diminuídos. Os níveis de expressão e a atividade de enzimas antioxidantes são conhecidamente baixos em ilhotas pancreáticas. A N-acetilcisteína (NAC) foi adicionada em cultura de células para prevenir o estresse oxidativo. Nessas condições, a NAC foi capaz de proteger as células INS-1E do estresse oxidativo induzido. Esses resultados sugerem que a exposição à polissacarose está associdada ao estresse oxidativo em células INS-1E e que a NAC foi capaz de prevenir a morte celular de células INS-1E expostas a ERO através do aumento intracelular de GSH. / Human pancreatic islet transplantation is considered a promising strategy to cure the cure Diabetes Mellitus type I. However, transplantation efficiency is dramatically affected by sub-optimum islet recovery in the isolation/purification procedure and islet viability after transplantation. Isolated pancreatic islets are obtained through collagenase perfusion and cell purification in a Ficoll gradient. Reactive oxygen species (ROS) play an important role during human pancreatic islet isolation and may contribute to the decrease in cell viability. The aim of this study was evaluated the response of human pancreatic islets during its isolation/purification and culture time. Activities of superoxide dismutase (SOD) and catalase as well as protein oxidation levels increased in most of analyzed samples, mainly during the Ficoll gradient islet purification step and further culture. Ficoll seems to be the critical step for ROS generation. Nevertheless, it was observed that donors characteristics (aging, cause of death, habits, etc.) and organ preservation conditions (ischemic time, preservation solution, etc.) may be related to our results. To minimize these variations, a physiological cellular model based on INS-1E cells was chosen. The antioxidant enzymes SOD, catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) activities as well the oxidative damage to proteins and lipids, reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, cellular viability and the protein levels of some enzymes responsible for apoptotic signaling like p38, JNK-1, ERK 1-2 and PI3-K upon exposure to polysucrose (1100 mg/mL), a similar of Ficoll, were determined. The SOD, catalase and GPx in samples exposed to polysucrose displayed hight activities. In all cultures, the activity of mitochondrial isoform of SOD (Mn-SOD) corresponds to 50% of total SOD activity. In the presence of polysucrose, the activity of Mn-SOD increased up to 80%. Lipids and protein oxidation levels were also increased and the GSH levels with the GR activity decreased. These results indicated that the exposure of INS-1E cells to polysucrose is associated with oxidative stress. However, the polysucrose exposure was not responsible for cell death although JNK-1, ERK1-2 and PI3-K levels showed hight levels but not p38, upon polysucrose exposure. The expression and activities of antioxidants enzymes are known to be very low in pancreatic islets. N-acetylcysteine (NAC) was added to the INS-1E cultures to prevent oxidative stress. Under these conditions, NAC was able to protect INS- 1E cells from induced oxidative damage by increasing intracellular GSH levels. Taken together, these results suggest that the exposure to polysucrose is related to the oxidative stress in INS-1E cells and NAC seems to be able to maintain cell viability.

La transplantation d’îlots pancréatiques offre un traitement potentielle pour le diabète de type 1 (T1DM). À ce jour, le succès mitigé de ce type de greffe est dû à plusieurs facteurs limitants comme le manque de revascularisation, la perte de la matrice extracellulaire (ECM) et le rejet par le système immunitaire du receveur. Dans les dernières années, l’utilisation de matrices tridimensionnelles (3D) et de bioréacteurs a amélioré le processus de transplantation et approfondi les connaissances sur le sujet. Le but de cette thèse est de mieux comprendre les effets des paramètres physiologiques (flux, concentration en oxygène dissous (D.O.) et pulsation) sur le tissu pancréatique dans un environnement 3D en utilisant un bioréacteur à perfusion. Le premier chapitre présente une revue de la littérature détaillant le pancréas, les maladies qui lui sont associées ainsi que les techniques permettant son étude in vitro et in vivo. L’utilisation de matrices 3D en recherche sur le diabète est discutée en profondeur tout en mettant l’emphase sur l’incorporation de molécules de la ECM. La revue souligne comment des matrices 3D testées en combinaison avec différents bioréacteurs ont permis de mieux comprendre et améliorer la culture de cellules pancréatiques. Une brève conclusion met en lumière les applications futures des bioréacteurs dans la recherche sur le diabète. La première étude de cette thèse traite de la culture de cellules de rat provenant d’insulinome (INS-1), encapsulées dans des matrices de fibrine en chambres de perfusion et cultivées dans un bioréacteur à perfusion. Un essai in situ de sécrétion d’insuline stimulée par le glucose fut développé pour comprendre les effets de la culture. Dans cette expérience, les effets bénéfiques des conditions contrôlées en bioréacteur à perfusion ont été démontrés et ont révélé une augmentation de l`indice de stimulation des cellules INS-1 avec le temps, une amélioration de la fonction GRIP, en plus d’une incidence moins élevée d’apoptose cellulaire en comparaison avec des témoins en culture statique, sans bioréacteur. Cette étude a été publiée dans la revue Biotechnology Progress. La deuxième étude décrit un design multifactoriel servant à l’identification des paramètres affectant des pancréas de rat dissociés mécaniquement, cultivés dans un bioréacteur à perfusion. Les effets uniques et combinés du flux, de la D.O. et de la pulsation ont été étudiés sur la culture de tissu pancréatique. Les conditions bénéfiques pour la culture en bioréacteur ont été identifiées. Le tissu pancréatique cultivé dans ces conditions bénéfiques a démontré une sécrétion d’insuline stimulée par le glucose, une plus grande activité métabolique, une coloration positive à l’insuline et au glucagon, des structures endothéliales multiples ainsi qu’un tissu plus intact en comparaison avec des cultures statiques cultivées en mode statique. Cette étude a été soumise à Biotechnology Progress. / Abstract : Transplantation of pancreatic islets offers a potential cure for type 1 diabetes mellitus (T1DM). To date, the success of such a graft has been mired by a number of limiting factors including lack of revascularisation, loss of native extracellular matrix (ECM), and graft rejection by the recipient’s immune system. In recent years, new ways to understand and improve this process have been explored using three-dimensional (3D) matrices and bioreactors. This thesis aims to further understand the important effect(s) physiological parameters (flow, dissolved oxygen concentration (D.O.) and pulsation) have on pancreatic tissue in a 3D environment using a perfusion bioreactor with defined geometries. The first chapter introduces a review of the literature detailing the native pancreas, its diseases, and how it is studied in vivo and in vitro. The use of 3D matrices in diabetes research is discussed with particular emphasis on the incorporation of ECM molecules. The review then highlights how 3D matrices have been used in combination with a host of different bioreactors to understand and improve pancreatic cell cultures. A brief conclusion about the future applications for the use of bioreactors in diabetes research is also discussed. The first experimental work comprises the culture of rat insulinoma cells (INS-1) encapsulated in fibrin matrices in perfusion chambers and cultured under perfusion bioreactor conditions. An in situ glucose-stimulated insulin secretion assay was then developed to monitor the culture over time. With this work, the beneficial effects of perfusion bioreactor conditions were shown and revealed increasing functionality (glucose-stimulated insulin secretion) of INS-1 cells over time, and a lower incidence of apoptosis when compared to static control cultures. This study was published in Biotechnology Progress. The second experimental work used a factorial design to identify process parameters affecting whole mechanically-disrupted rat pancreata in a perfusion bioreactor. Here, the singular and combinational effects of flow, dissolved oxygen concentration and pulsation were assessed on the outcome of pancreatic tissue. Beneficial bioreactor conditions were identified. Mechanically-disrupted rat pancreata cultured under these beneficial bioreactor conditions showed glucose-stimulated insulin secretion, higher metabolic activity, insulin- and glucagon-positive staining, extensive endothelial structures, and overall intact tissue when compared to static cultures. This study has been submitted to Biotechnology Progress.

Diabetes Mellitus is a group of complex and heterogeneous metabolic disorders characterized by hyperglycemia. Even though the condition has been extensively studied, its causes and complex pathologies are still not fully understood. The occurring damage to the pancreatic islets is strikingly heterogeneous. In type 1 diabetes, the insulin producing beta cells are all destroyed within some islets, and similarly in type 2 diabetes, some islets may be severely affected by amyloid. At the same time other islets, in the near vicinity of the ones that are affected by disease, may appear fully normal in both diseases. Little is known about this heterogeneity in susceptibility to disease between pancreatic islets. This thesis examines the physiological and pathophysiological characteristics of islet subpopulations. Two subpopulations of islets were studied; one constituting highly vascularized islets with superior beta cell functionality, and one of low-oxygenated islets with low metabolic activity. The highly functional islets were found to be more susceptible to cellular stress both in vitro and in vivo, and developed more islet amyloid when metabolically challenged. Highly functional islets preferentially had a direct venous drainage, facilitating the distribution of islet hormones to the peripheral tissues. Further, these islets had an increased capacity for insulin secretion at low glucose levels, a response that was observed abolished in patients with recent onset type 1 diabetes.  The second investigated islet subpopulation, low-oxygenated islets, was found to be an over time stable subpopulation of islets with low vascular density and beta cell proliferation. In summary, two subpopulations of islets can be identified in the pancreas based on dissimilarities in vascular support and blood flow. These subpopulations appear to have different physiological functions of importance for the maintenance of glucose homeostasis. However, they also seem to differ in vulnerability, and a preferential death of the highly functional islets may accelerate the progression of both type 1 and type 2 diabetes.

O transplante de ilhotas microencapsuladas constitui uma alternativa terapêutica interessante para o Diabetes Mellitus tipo 1, permitindo um melhor controle glicêmico e eliminando a necessidade de imunossupressão. Entretanto, a manutenção a longo prazo da viabilidade das células-&#946; ainda é um desafio. No isolamento, a perda da matriz extracelular e as condições hipóxicas subsequentes afetam decisivamente a sobrevivência e funcionalidade das ilhotas. Objetivo Para diminuir o estresse sobre o enxerto, levando a um sucesso prolongado do transplante, propôs-se a adição de perfluorocarbono (PFC) ou laminina (LN), moléculas associadas respectivamente à oxigenação e interações célula-célula, ao biomaterial baseado em alginato, Biodritina, adequado ao encapsulamento celular. Metodologia Para testar a estabilidade das formulações PFC-Biodritina e LN-Biodritina, microcápsulas foram submetidas a diferentes estresses (rotacional, osmótico, temperatura e cultura) por 7 e 30 dias. A pureza do biomaterial foi avaliada pela coincubação com macrófagos murinos RAW264.7, por 3, 9 e 24h, quando a ativação dos macrófagos foi observada pela expressão gênica de IL- 1&#946; e TNF&#945;. Microcápsulas implantadas i.p. em camundongos foram recuperadas após 7 ou 30 dias, para análises de biocompatibilidade. A expressão de níveis de mRNA (bax, bad, bcl-2, bcl-XL, xiap, caspase 3, mcp1/ccl2, hsp70, ldh, insulina 1 e 2), proteínas (Bax, Bcl-XL e Xiap) e a atividade de Caspase3 foram avaliadas em ilhotas microencapsuladas com PFC- e LN-Biodritina, após cultura de 48h em condições de normóxia e hipóxia (<2% O2). Camundongos diabéticos foram transplantados com ilhotas encapsuladas nas diferentes formulações e os animais foram monitorados pelas variações de massa corporal, glicêmicas e pela funcionalidade do enxerto (TOTGs). As ilhotas foram recuperadas de animais normo ou hiperglicêmicos e uma análise de biocompatibilidade das cápsulas foi realizada, assim como a avaliação funcional das células-&#946;. Após o explante, a glicemia dos animais normoglicêmicos foi monitorada para se atestar a eficiência das ilhotas transplantadas. Resultados Microcápsulas de PFC- e LN-Biodritina são tão estáveis e biocompatíveis quanto as de Biodritina. Para ilhotas encapsuladas em ambos os materiais, em normóxia ou hipóxia, observou-se uma modulação gênica que sugere proteção contra apoptose. Adicionalmente, encontrou-se uma diminuição na expressão de genes indicadores de estresse (mcp1, hsp70). Uma diminuição nos níveis de mRNA de ldh foi vista para PFC-Biodritina, mas o oposto foi encontrado para LN-Biodritina. As diferenças encontradas na expressão proteica sugerem o mesmo padrão anti-apoptótico. Caspase3 não foi modulada por nenhum biomaterial. Nos experimentos de transplante, apenas LN-Biodritina levou reversão prolongada do diabetes, com 60% dos animais normoglicêmicos, 198 dias pós-cirurgia, comparado a 9% do grupo Biodritina. O TOTG demonstrou que camundongos transplantados com ilhotas encapsuladas secretaram mais insulina do que controles, 60 (LN-Biodritina) ou 100 (PFC- e LN-Biodritina) dias pós-cirurgia. O explante restabeleceu a hiperglicemia nos camundongos. Microcápsulas recuperadas de animais hiperglicêmicos apresentavam uma extensa adesão celular. Testes de secreção de insulina in vitro demonstraram que somente ilhotas do grupo normoglicêmico responderam às variações da concentração de glicose. Conclusão A adição de moléculas bioativas à Biodritina é capaz de diminuir o estresse em ilhotas isoladas e tem o potencial de melhorar a terapia pelo transplante de ilhotas. / Transplantation of microencapsulated islets represents an attractive therapeutical approach to treat type 1 Diabetes Mellitus, accounting for an improved glycemic control and the abolishment of immunosuppressive therapies. However, maintenance of long-term &#946;-cell viability remains a major problem. During islet isolation, the loss of extracellular matrix interactions and the hypoxic conditions thereafter dramatically affect &#946;-cell survival and function. Objective To lessen the burden of islet stress and achieve a better outcome in islet transplantation we tested the addition of perfluorocarbon (PFC) or laminin (LN), molecules associated respectively with oxygenation and cell-cell interaction, to Biodritin, an alginate-based material suitable for cell microencapsulation. Methodology To test the stability of PFC-Biodritin and LN-Biodritin composites, microcapsules were subjected to different stresses (rotational, osmotic, temperature and culture) for 7 and 30 days. To assess biomaterial purity microcapsules were co-incubated with RAW264.7 murine macrophage cell line for 3, 9 and 24h and macrophage activation was detected through mRNA levels of IL-1&#946; and TNF&#945;. Microcapsules were implanted i.p. in mice and retrieved after 7 or 30 days, for biocompatibility analyses. Gene expression at mRNA (bax, bad, bcl-2, bcl-XL, xiap, caspase 3, mcp1/ccl2, hsp70, ldh, insulin 1 and 2) and protein (Bax, Bcl-XL and Xiap) levels, together with Caspase3 activity, were evaluated in islets microencapsulated in PFC- or LN-Biodritin, upon culturing for 48h in normoxic or hypoxic (<2% O2) conditions. Diabetic mice were transplanted with PFC- or LN-Biodritin microencapsulated islets, followed by assessments of body weight, glycemia and graft function by oral glucose tolerance tests (OGTTs). Microencapsulated islets were retrieved from normoglycemic or hyperglycemic mice and biocompatibility analyses of the beads together with a functional assessment of the graft followed. After graft removal, normoglycemic animals had their glycemias monitored to attest the efficacy of the transplanted islets. Results PFC- and LN-Biodritin microcapsules were as stable and biocompatible as Biodritin. For both biomaterials in normoxia and hypoxia a modulation in gene expression was observed in islets associated with a protection against apoptosis. Also, a decreased expression of stress-related genes (mcp1, hsp70) was evidenced. ldh mRNA levels were down-regulated in PFC-Biodritin microencapsulated islets but upregulated in the presence of LN. Increased levels of insulin mRNA were observed. The differences seen in protein expression indicated the same anti-apoptotic pattern. Caspase3 activity was not different between groups. Concerning diabetes reversal experiments, only mice transplanted with LN-Biodritin microencapsulated islets presented a better outcome, with 60% remaining euglycemic at 198 days post-surgery, compared with 9% for the Biodritin group. OGTT showed that mice transplanted with encapsulated islets secreted more insulin than normal mice, 60 (LN-Biodritin) or 100 days (PFC- and LN-Biodritina) posttransplant. Hyperglycemia was achieved after the retrieval of microcapsules showing graft efficacy. Retrieved microcapsules revealed an extensive overgrowth in most beads from hyperglycemic mice. A static glucose stimulated insulin secretion test revealed that only islets from normoglycemic subjects were able to secrete insulin according to glucose concentration. Conclusion- The addition of bioactive molecules to Biodritin may lessen the stress of isolated islets and have the potential to improve islet transplantation therapy.

L’ischémie est une des problématiques majeurs de la transplantation, elle intervient au moment de la déconnexion d’un organe du système vasculaire et dure jusqu’à sa reconnexion chez le receveur. Elle provoque une diminution de la qualité des greffons et seulement un-tiers des pancréas éligibles aboutissent à une transplantation. Ce travail de thèse avait pour objectif d’améliorer la qualité des greffons en préparant les pancréas à subir l’ischémie par le préconditionnement et à améliorer leur préservation après avoir identifié les voies impliquées dans l’ischémie. Des changements métaboliques majeurs au niveau de l’organe ont été mis en évidence dépendant du temps d’ischémie, impliquant des dommages sur la partie endocrine du pancréas, les îlots. Ceci a été empêché par le préconditionnement du pancréas à l’ischémie activant les défenses antioxydantes et par l’utilisation de l’hémoglobine M101 issue de ver marin apportant de l’oxygène au cours de la préservation des pancréas de rat et humains. Ainsi, il est possible d’améliorer la qualité des greffons en agissant en amont du prélèvement et au cours de la préservation, ceci permettant une meilleure fonction. / Ischemia is one of the major problems of transplantation, it occurs at the time of disconnection of an organ of the vascular system and lasts until its reconnection in the recipient. It causes a decrease in graft quality and only one-third of the eligible pancreases results in a transplant. This work aimed to improve the quality of the grafts in preparing the pancreas to undergo ischemia preconditioning and improve their preservation after identifying the pathways involved in ischemia. Major metabolic changes in the organ have been demonstrated that appeared to ischemia time dependent enhancing damages to the endocrine part of the pancreas, the islets. This was prevented by the preconditioning of the pancreas to ischemia activating the antioxidant defenses and by the use of the marine worm M101 hemoglobin providing oxygen during the preservation of rat pancreas and humans. Thus, it is possible to improve the quality of the grafts by acting upstream of the sample and during the preservation, this allowing a better function.

No description available.

O Diabetes mellitus (DM) do tipo 1 é uma doença causada pela destruição, por mecanismo auto-imune, das células beta das ilhotas pancreáticas, produtoras de insulina. O tratamento convencional da doença é realizado por meio de injeções diárias de insulina exógena. O transplante de ilhotas pancreáticas inclui-se, atualmente, como uma das alternativas terapêuticas à insulinoterapia. Entretanto, para atingir a insulino-independência, é necessário transplantar um grande número de ilhotas por paciente. O conhecimento do mecanismo de proliferação das células beta pode possibilitar a realização do transplante a partir da expansão celular ex vivo. A glicose é um dos principais indutores da proliferação de células beta. Neste trabalho, foi estabelecida e executada a tecnologia de isolamento e purificação de ilhotas pancreáticas humanas, visando sua estimulação com glicose. Para identificar genes regulados por glicose nestas ilhotas, foi utilizada a técnica de hibridização subtrativa SSH, associada ao rastreamento da biblioteca através de macroarranjos de DNA. Num primeiro rastreamento, foram identificados dois fragmentos gênicos induzidos pela glicose. Um destes apresentou homologia com uma proteína hipotética humana de função desconhecida e o segundo com o receptor de polipetídeo pancreático. Este trabalho permitiu a identificação de novos genes regulados pela glicose em ilhotas pancreáticas humanas, os quais podem estar relacionados à proliferação celular deste tecido. / Type 1 Diabetes mellitus (T1DM) is caused by autoimmune destruction of the insulin-producing pancreatic islet b-cells. Treatment is generally approached by daily subcutaneous injections of exogenous insulin. Nowadays, pancreatic islet transplantation is considered as an effective alternative treatment to insulin therapy. However, in order to reach insulin-independence, a large number of islets is required for each patient. Knowledge of the mechanisms regulating islet b-cell proliferation may allow ex-vivo b-cell expansion prior to transplant. Glucose is considered one of the main inducers of islet b-cells proliferation. We established and executed the technology of human islet isolation and purification. The islets were then stimulated in culture with glucose. In order to identify glucose-regulated genes in cultured human islets, we utilized the suppression subtractive hybridization (SSH) method, followed by cDNA library screening by DNA macroarrays. Preliminary screening allowed us to isolate two cDNAs displaying glucose regulation, one of which is similar to a human hypothetical protein of unknown function and the other shows similarity to the pancreatic polypeptide receptor. This work allowed identification of glucose-regulated genes in human pancreatic islets, which may be related to cell proliferation in this tissue.

The aim of the study was to examine the immune response and different immunoprotective strategies in experimental type 1 diabetes mellitus. The autoimmune destruction of the insulin-producing pancreatic β-cells that leads to type 1 diabetes is complex and incompletely understood. Activated immune cells infiltrate the pancreatic islets at an early stage of the disease, and they produce and release cytokines, which may contribute to β-cell dysfunction and death. Several immunomodulatory agents with different mechanisms have recently been developed in order to suppress cytokine function such as MDL 201, 449A, a novel transcriptional inhibitor of TNF-α. At least in rodent β-cells, many of the toxic actions of cytokines depend on the synthesis of nitric oxide (NO). Aminoguanidine (AG), an inhibitor of NO formation, might therefore be an interesting compound for prevention of type 1 diabetes. Another substance that could influence the course of events leading to this disease is the pituitary hormone prolactin (PRL), since it has the ability to activate different immune cells. We have studied the effects of AG, PRL and MDL 201, 449A on the development of hyperglycaemia and pancreatic insulitis in multiple low dose streptozotocin induced autoimmune diabetes in mice. The natural course after syngeneic islet transplantation of pancreatic islets in NOD mice, a model of type 1 diabetes mellitus was also investigated. AG and PRL were also studied in vitro on cultured isolated rodent pancreatic islets. We suggest that the insulin-producing cells are specifically targeted by the inflammatory response after syngeneic islet transplantation in type 1 diabetic mice. Our data do not exclude a role for NO in type 1 diabetes, but it raises concerns about the use of AG as a therapeutic agent since an increased mortality and no decline in diabetes frequency was observed. AG did not seem to be directly harmful to β-cell function, but it could affect pancreatic and islet blood flows. PRL and MDL 201, 449A could both counteract hyperglycaemia and insulitis in the early phase of autoimmune diabetes.

De l’isolement des îlots pancréatiques à leur implantation, l’inflammation est omniprésente au cours de la transplantation d’îlots pancréatiques. Le maintien d’une inflammation contrôlée est essentiel pour préserver la survie et la fonctionnalité du greffon à court et long terme. L’objectif de ce travail de thèse est d’identifier précisément les mécanismes inflammatoires à l’origine de la perte précoce des îlots et de déterminer des cibles thérapeutiques pour limiter ces réactions inflammatoires.Nous avons ainsi démontré que les conditions de culture induisent des réactions à l’origine du développement d’un phénotype pro-inflammatoire et pro-oxydant propre à l’îlot. Cette induction se caractérise par une élévation de la sécrétion de cytokines, de chimiokines pro-inflammatoires, une activation des voies de l’inflammation Toll-like récepteurs (TLRs)-dépendantes et une génération d’espèces réactives de l’oxygène (ROS). Toutefois, ce processus peut être prévenu par l’activation de l’Hème oxygénase-1 (HO-1), une enzyme anti-oxydante et anti-inflammatoire.Par l’étude des réactions inflammatoires sur un modèle animal de transplantation mimant les conditions de transplantation humaine, nous avons démontré qu’un changement des médiateurs plasmatiques de l’inflammation et du protéome hépatique s’opère 12 heures après transplantation. De plus, ces résultats sont associés à une infiltration des îlots par les cellules immunitaires qui s’organise 12 heures après transplantation. Nous avons également établi le rôle anti-inflammatoire de la rapamycine (une drogue immunomodulatrice) sur les îlots et les macrophages in vitro. Nous avons ainsi démontré que l’usage de la rapamycine avec la mise en place d’un pré-traitement des îlots et du receveur avant la greffe serait envisageable. Ces travaux ont permis de caractériser les mécanismes inflammatoires mis en oeuvre immédiatement avant et après transplantation. Ainsi, ces données offrent de nouvelles pistes thérapeutiques susceptibles de prévenir et/ou limiter l’inflammation au cours de la transplantation d’îlots pancréatiques. / From isolation of pancreatic islets to their implantation, the inflammation is ubiquitous in the pancreatic islet transplantation. Maintaining a controlled inflammation is essential to preserve the survival of the graft and the functionality in the short and long term. The objective of this work is to identify precisely the inflammatory mechanisms behind the early loss of islets and identify therapeutic targets to reduce these inflammatory reactions. We have demonstrated that culture conditions induce reactions causing the development of a specific proinflammatory and pro-oxydant phenotype islet. This induction is characterized by an increase in the secretion of cytokines, chemokines pro-inflammatory activation pathways of inflammation Toll-like receptors (TLRs) -dependent and generation of reactive oxygen species (ROS). However, this process can be prevented by the activation of Heme oxygenase-1 (HO-1), an antioxidant and anti-inflammatory enzyme.By studying the inflammatory responses in an animal model of transplantation mimicking the conditions of human transplantation, we demonstrated that a change of plasma mediators of inflammation and liver proteome occurs 12 hours after transplantation. Furthermore, these results are associated with infiltration of the islets by immune cells which organizes 12 hours after transplantation. We also determined the anti-inflammatory role of rapamycin (an immunomodulatory drug) on the islets and macrophages in vitro. We have thus demonstrated that the use of rapamycin with the establishment of a pre-treatment of islets and recipient before transplantation could be considered. These studies have characterized the inflammatory mechanisms implemented immediately before and after transplantation. Thus, these data provide new therapeutic approaches that can prevent and / or reduce inflammation during pancreatic islet transplantation