Currently, the transplantation of islets of Langerhans is a viable means to maintain control of blood sugar levels and reduce the risk of hypoglycemia in defined populations with brittle type I diabetes mellitus or those requiring pancreatectomy. However, the process of islet isolation is highly variable and not all isolations result in islet numbers or quality suitable for transplantation. This thesis aimed to improve transplantation success through optimization and standardization of the isolation process and to identify pretransplant variables associated with early islet engraftment. A previously disregarded enzyme activity, tryptic-like activity (TLA), has been identified to influence pancreas digestion efficiency and islet isolation success in both the preclinical and clinical situations. For human pancreases, islet isolation success rates improved from 0% in the lowest TLA group to over 50% in the highest TLA groups without affecting islet quality. These findings should help standardize evaluation of enzymes for clinical islet isolation. A closed, automated, pump-made gradient system was compared to the open, manual method for islet separation. No differences were observed in expected gradient volumes, islet yields or total purities between the two methods. The pump-made gradient system successfully removed manual influences on density gradient production while fulfilling regulatory requirements for closed system processing. Islet quantification was evaluated with computer-assisted digital imaging analysis (DIA) and a semi-closed assessment system. By using the DIA system method, which measures islet purity and pellet volume instead of manual counting methods, variation in islet counts and purity reduced by almost half. By using a transplant outcome measurement of C-peptide adjusted by blood glucose and creatinine, we identified four pretransplant factors that affect early transplant outcome. Of the four factors, one was related to the organ transport time, one to function of the islets, and two to the transplanted tissue volume. When these four factors were put into a predictive model, it accounted for about 40% of the transplant outcome. The work contained in this thesis identifies and optimizes a number of critical elements related to islet isolation and transplantation protocols.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 2 uppsatser.

<p>Type 1 Diabetes Mellitus (T1D), a chronic disease affecting over 1.5 million Americans, is characterized by the autoimmune destruction of insulin-producing β-cells within pancreatic islets. Islet/β-cell replacement therapies, where replenishable β-cell sources are implanted within protective microenvironments, have the potential to provide a long-term solution for individuals with T1D by restoring glucose-sensitive, insulin release and overall glycemic control. However, most conventional encapsulation materials elicit an immune reaction, known as a foreign body response (FBR), which compromises β-cell health and function. In this dissertation, we designed and evaluated various formulations of a polymerizable collagen, namely type I oligomeric collagen (Oligomer), as encapsulation materials for minimally invasive, subcutaneous delivery of replacement β-cells. Preclinical validation in chemically-induced diabetic mice demonstrated rapid (within 24 hours) reversal of diabetes for beyond 90 days with no signs of rejection or FBR after subcutaneous delivery of both allogeneic and xenogeneic (rat) islets. To further define this uncommon mechanism of protection, the tissue response to Oligomer, in comparison to commercial synthetic and collagen-based materials, was evaluated following subcutaneous implantation within rats, a well-established biocompatibility model. Histological and transcriptomics analyses were used to define the immune response at both cellular and molecular levels. Interestingly, Oligomer showed minimal and transient activation of innate immune cells similar to the sham surgical control, with no evidence of foreign body giant cell formation, inflammatory-mediated bioresorption, or fibrosis. Overall, this work evaluates preclinical efficacy and demonstrates mechanistic understanding of immune tolerance for Oligomer materials for β-cell replacement therapy and other regenerative medicine applications.</p>

Résumé : Durant les dernières années, l’encapsulation d’îlots pancréatiques endocriniens a reçu une grande attention parce qu’elle pourrait constituer une solution pour diminuer les taux d'échecs des transplantations. Dans le contexte de la perte de la matrice extracellulaire (MEC) native des îlots lors de leur isolation et le rejet de greffes par le système immunitaire du receveur, cette thèse vise à améliorer la compréhension des interactions entre la MEC et les cellules des îlots pancréatiques endocriniens ainsi qu’à étudier les effets de stress apoptotiques associés à des éléments du système immunitaire sur la survie et les fonctions des îlots. Ces études pourraient permettre de raffiner notre compréhension des mécanismes associés au rejet des greffes d'îlots de Langerhans. Dans cette thèse, le premier chapitre constitue une revue de la littérature permettant de mettre en lumière les rôles réciproques de la MEC dans l'action des cellules immunitaires et l'influence de ces rôles sur le diabète de type 1 (DT1) et sur la transplantation d'îlots. Ce premier chapitre a été publié dans la revue Pathologie Biologie. Le premier travail expérimental comprend la culture de cellules d'insulinomes de rat (INS-1) sur des surfaces composées de carboxyméthyl dextrane (CMD) recouvertes de fibronectine, RGD ou YIGSR, un peptide synthétique de la laminine. Dans cette étude, l'effet bénéfique d’éléments de la MEC sur ces cellules productrices d'insuline a été démontré. Les cellules INS-1 ont davantage proliféré sur ces surfaces et sécrétaient plus d’insuline que les cellules INS-1 cultivées sur les surfaces contrôle de CMD, CMD+RGE et dans les plaques à multi-puits de polystyrène vendues pour la culture tissulaire (TCPS). Cette première étude a été publiée dans Acta Biomaterialia. La deuxième étude expérimentale avait pour objectif d’étudier l’effet protecteur de gels de fibrine pour enrober des îlots pancréatiques endocriniens isolés de jeunes porcs et exposés à deux concentrations de peroxyde d'hydrogène (H[indice inférieur 2]O[indice inférieur 2]). L’enrobage dans la fibrine a permis de réduire l'apoptose chez les cellules des îlots et d’améliorer la sécrétion d'insuline par ceux-ci lorsque les résultats étaient comparés à ceux des îlots non-enrobés. Ce travail a été publié dans la revue Islets. Dans la troisième étude expérimentale, des îlots porcins étaient enrobés dans des gels de fibrine et d'alginate et exposés à des monocytes humains pour comparer l’effet de l’enrobage par ces deux matériaux sur la survie et les fonctions des îlots. Les monocytes sécrétaient des concentrations importantes de cytokines TNFα, IL-6, IL-1β en réponse à la fibrine seule et aux îlots. Les cellules des îlots enrobés dans les gels de fibrine et d'alginate étaient moins apoptotiques et sécrétaient plus d'insuline que leurs contrôles respectifs non-enrobés. Cette étude a été acceptée dans la revue Pathologie Biologie. // Abstract : In recent years, the encapsulation of endocrine pancreatic islets has received enhanced attention as it might constitute a solution for islet transplantation failure. In the context of the loss of the native islet extracellular matrix (ECM) and graft rejection by the recipient’s immune system, this thesis aims to improve the understanding of ECM-islet cell interactions and immune system-related implications in islet survival and function in the context of type 1 diabetes mellitus (T1DM) and islet graft rejection. In the first chapter, a literature review introduces the reciprocal roles of the ECM in immune cell action and the influence of these interactions on T1DM and islet transplantation. The most important ECM components are discussed followed by an overview of immune cells and their possible implication in diabetes. Immune cell integrins and cytokines and their communication with and influence on ECM are highlighted, concluding in a brief discussion of the significance of these interactions for islet transplantation and encapsulation. This review has been accepted for publication by Pathologie Biologie. The first experimental work comprises the culture of rat insulinoma cells (INS-1) on welldefined low-fouling carboxymethyl-dextran (CMD) surfaces covalently grafted with fibronectin, RGD and YIGSR, a synthetic laminin peptide, resulting in higher cell proliferation and insulin secretion of INS-1 cells when compared to the controls CMD, CMD+RGE and tissue culture polystyrene (TCPS) plates. With this work, the beneficial effect of ECM cues on insulin-producing cells was proven. This study has been published in Acta Biomaterialia. The second experimental work aimed to study the effect of fibrin gels when used to embed endocrine pancreatic islets isolated from young pigs and exposed to hydrogen peroxide (H[subscript 2]O[subscript 2]). Fibrin-embedded islets showed less apoptosis and higher relative insulin secretion than islets on TCPS, verifying the protective effect of fibrin towards islets. This study has been published in Islets. In the third experimental study, porcine islets were encapsulated in fibrin and alginate gels and exposed to human monocytes to compare the two materials and to further investigate the immune protective properties of fibrin and alginate. Monocytes secreted high concentrations of TNFα, IL-6, and IL-1β in response to fibrin, but at the same time islets in both fibrin and alginate gels were less apoptotic and secreted more insulin then their TCPS controls. This study has been submitted to Pathologie Biologie.

<p>The presence of protein amyloid in pancreas and its association to diabetes was first described 100 years ago in 1901, but was not identified as Islet Amyloid Polypeptide (IAPP) until 1986. The aim of the present work was to determine the role of the beta cell hormone, IAPP, in normal pancreatic islet physiology and during early disturbances of islet function.</p><p>Intra-islet peptides, i.e. chromogranin peptides and an extra-islet peptide, i.e. leptin, were studied to identify possible endogenous regulators of IAPP and insulin secretion. Chromogranin-B, but not chromogranin-A or pancreastatin, had the ability to inhibit islet IAPP and insulin release, suggesting that chromogranin-B may serve as an autocrine regulator of IAPP and insulin secretion. </p><p>Leptin had a more potent effect on IAPP secretion than on insulin secretion, which was dissociated from effects on islet glucose metabolism. Glucose oxidation rates were increased at physiological leptin concentrations, whereas higher leptin concentrations showed an inhibitory effect and chronically high leptin concentrations had no effect.</p><p>Female NOD mice were studied to investigate the release of IAPP in the progression to type 1 diabetes. The release of IAPP was lower than that of insulin from immune cell infiltrated islets, indicating preferential insulin release during the early course of the disease. </p><p>IAPP is expressed at an early embryonic stage. The effect of IAPP on cell proliferation in neonatal rat islets was studied in the search for a physiological role of IAPP. IAPP concentrations of (1-1000) nM stimulated neonatal islet cell proliferation mostly in beta cells and to a lesser extent in alpha cells. IAPP did not have any marked effect on the islet cell death frequency. These data indicate a role for IAPP as a potential regulator of beta cell proliferation in neonatal pancreatic islet.</p><p>It is concluded that IAPP may be involved in regulation of pancreatic beta cell function both in fetal and adult life.</p>

The presence of protein amyloid in pancreas and its association to diabetes was first described 100 years ago in 1901, but was not identified as Islet Amyloid Polypeptide (IAPP) until 1986. The aim of the present work was to determine the role of the beta cell hormone, IAPP, in normal pancreatic islet physiology and during early disturbances of islet function. Intra-islet peptides, i.e. chromogranin peptides and an extra-islet peptide, i.e. leptin, were studied to identify possible endogenous regulators of IAPP and insulin secretion. Chromogranin-B, but not chromogranin-A or pancreastatin, had the ability to inhibit islet IAPP and insulin release, suggesting that chromogranin-B may serve as an autocrine regulator of IAPP and insulin secretion. Leptin had a more potent effect on IAPP secretion than on insulin secretion, which was dissociated from effects on islet glucose metabolism. Glucose oxidation rates were increased at physiological leptin concentrations, whereas higher leptin concentrations showed an inhibitory effect and chronically high leptin concentrations had no effect. Female NOD mice were studied to investigate the release of IAPP in the progression to type 1 diabetes. The release of IAPP was lower than that of insulin from immune cell infiltrated islets, indicating preferential insulin release during the early course of the disease. IAPP is expressed at an early embryonic stage. The effect of IAPP on cell proliferation in neonatal rat islets was studied in the search for a physiological role of IAPP. IAPP concentrations of (1-1000) nM stimulated neonatal islet cell proliferation mostly in beta cells and to a lesser extent in alpha cells. IAPP did not have any marked effect on the islet cell death frequency. These data indicate a role for IAPP as a potential regulator of beta cell proliferation in neonatal pancreatic islet. It is concluded that IAPP may be involved in regulation of pancreatic beta cell function both in fetal and adult life.

De nombreuses protéines associées aux maladies neurodégénératives humaines sont intrinsèquement désordonnées. Ce sont des protéines qui sont dépourvues de structure tertiaire ou secondaire stable dans des conditions physiologiques. Plus précisément, les protéines intrinsèquement désordonnées (IDPs) subissent diverses changements conformationnels entre la pelote aléatoire, des conformations hélicoïdales et des structures en feuillet-β, ces deux dernières étant généralement impliquées dans la reconnaissance protéine-protéine. Parmi une vingtaine de peptides amyloïdogènes connus liés aux maladies dégénératives humaines, notre étude porte sur deux protéines désordonnées: le peptide Amyloïde-β (Aβ) associé à la maladie d'Alzheimer et l'Islet Amyloid Polypeptide (IAPP) impliqué dans le diabète de type II. Aβ possède deux alloformes courants de 40 et 42 résidus, tandis que IAPP est une hormone peptidique de 37 résidus. Les agrégats de Aβ sont toxiques pour les cellules du cerveau, tandis que la fibrillisation de IAPP affecte les cellules-β du pancréas. Le mécanisme d'agrégation de ces deux peptides reste encore mal connu, mais il a été proposé qu’en solution, ces peptides visitent différentes conformations, l'une d'entre elles étant riche en feuillets-β. Cela conduirait à l’oligomérisation de ces peptides, par le biais d’interactions feuillet-β / feuillet-β et, éventuellement, à la formation de fibrilles. Le but de notre étude est de mieux caractériser la dynamique conformationnelle de ces deux peptides, dans leur forme monomérique et oligomérique. Comprendre les premières étapes de leur agrégation est crucial pour le développement de nouvelles molécules thérapeutiques efficaces contre ces protéines amyloïdes. / Many proteins associated with human neurodegenerative diseases are intrinsically disordered. They are proteins which lack stable tertiary or secondary structure under physiological conditions. More specifically, intrinsically disordered proteins (IDPs) undergo various structural conversions between random coil, helical conformations and β-strand structures, these two latter being generally involved in protein-protein recognition. Among about twenty known amyloidogenic peptides related to human degenerative diseases, we focus our study on two disordered proteins: the Amyloid-β peptide (Aβ) associated to the Alzheimer’s disease and the Islet Amyloid Polypeptide (IAPP) involved in type II diabetes. Aβ has two common alloforms of 40 and 42 residues in length, meanwhile IAPP is a 37-residues peptide hormone. Aggregates of Aβ are toxic to the brain cells, meanwhile IAPP fibrillization affects the pancreatic β-cells. The aggregation mechanism of these two peptides is not known in detail, but it was proposed that in solution, these peptides visit various conformations, one of them being rich in β-strands. This would lead to peptide oligomerization, through β-strand / β-strand interactions and eventually to the fibril formation. The aim of our study is to provide insights into the conformational dynamics of these two peptides in monomeric and oligomeric forms. Understanding the early steps of their aggregation is crucial for the development of new effective therapeutic molecules against these amyloid proteins.De nombreuses protéines associées aux maladies neurodégénératives humaines sont intrinsèquement désordonnées. Ce sont des protéines qui sont dépourvues de structure tertiaire ou secondaire stable dans des conditions physiologiques. Plus précisément, les protéines intrinsèquement désordonnées (IDPs) subissent diverses changements conformationnels entre la pelote aléatoire, des conformations hélicoïdales et des structures en feuillet-β, ces deux dernières étant généralement impliquées dans la reconnaissance protéine-protéine. Parmi une vingtaine de peptides amyloïdogènes connus liés aux maladies dégénératives humaines, notre étude porte sur deux protéines désordonnées: le peptide Amyloïde-β (Aβ) associé à la maladie d'Alzheimer et l'Islet Amyloid Polypeptide (IAPP) impliqué dans le diabète de type II. Aβ possède deux alloformes courants de 40 et 42 résidus, tandis que IAPP est une hormone peptidique de 37 résidus. Les agrégats de Aβ sont toxiques pour les cellules du cerveau, tandis que la fibrillisation de IAPP affecte les cellules-β du pancréas. Le mécanisme d'agrégation de ces deux peptides reste encore mal connu, mais il a été proposé qu’en solution, ces peptides visitent différentes conformations, l'une d'entre elles étant riche en feuillets-β. Cela conduirait à l’oligomérisation de ces peptides, par le biais d’interactions feuillet-β / feuillet-β et, éventuellement, à la formation de fibrilles. Le but de notre étude est de mieux caractériser la dynamique conformationnelle de ces deux peptides, dans leur forme monomérique et oligomérique. Comprendre les premières étapes de leur agrégation est crucial pour le développement de nouvelles molécules thérapeutiques efficaces contre ces protéines amyloïdes.

No description available.

No description available.

Genome-wide association studies (GWAS) have made substantial progress in implicating genomic regions in type 2 diabetes (T2D) susceptibility. Whilst attributing causal mechanisms to loci has proved non trivial, these studies have provided insights into the genetic architecture underlying the disease. GWAS findings indicate a causal role for gene regulatory processes, and suggest that pancreatic beta-cells play a pivotal role in mediating common T2D association. Work presented in this thesis therefore sought to generate novel regulatory annotations from human islets, and to assess whether T2D-associated loci can be accurately fine-mapped using statistical approaches, with the aim of improving understanding of causal mechanisms underlying these associations through integration of the two approaches. Using small RNA sequencing in human islets and enriched beta-cell populations (both n=3) and mRNA sequencing in a large number of human islets (n=130), I increased the number of available human islet annotations. These studies identified high or islet-specific expression in many micro RNAs (miRNAs) without previously known roles in human islets. It also provided the largest study of quantitative trait loci (eQTLs) and allele-specific expression (ASE) in human islets to date, identifying significant eQTLs for 1,636 genes and significant ASE at 8,754 genes. There was enrichment of active islet chromatin, compared to other tissues, at the best eQTL variant for each gene, but also substantial sharing of significant eQTLs between islets and other tissues. Simulations were used to assess the utility of fine-mapping approaches for refining common disease-associated loci to smaller intervals or sets of variants likely to include the causal variant. The results demonstrated that fine-mapping can indeed refine these loci to sets or intervals of a size more amenable to functional follow-up or focussed intersection with high quality annotations. Furthermore, using an approximated Bayesian approach, I was able to refine twenty-one of the known common T2D-associated loci. Finally, using the newly generated annotations, I demonstrated enrichment of T2D association signal for regulatory RNA annotations (islet lncRNAs and miRNA target gene sets). I also identified examples in which these types of annotation overlap common and rare variation suggestive of a role in T2D pathogenesis. Using further islet annotations, I also uncovered potential causal mechanisms at four of the twentyone fine-mapped common T2D loci. These data therefore provide many novel islet regulatory annotations that can be intersected with T2D genetics, and provide a first example of how such an approach can lead to novel potential causal mechanisms underlying association loci.