Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

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

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.

The islets of Langerhans are small and delicate spheroid organs scattered in the pancreas responsible for insulin production. Transplantation of isolated islets is a beneficial therapy for patients with a severe form of type 1 diabetes. The islets, which normally are richly vascularized in the pancreas, are completely disconnected from the vascular support by the enzymatic digestion during the isolation procedure. Islet viability is affected throughout all steps in this process, from donor death and isolation of islets to culturing and the transplantation process itself. In this thesis a novel strategy to promote islet survival and to re-establish islet vasculature is presented. We show endogenous expression of 51 different genes related to inflammation in cultured islets. Among these genes high expression of MCP-1, MIF, VEGF, thymosin b-10 and IL-8, IL-1β, IL-5R-a, IFN-γ antagonist were found in all donors during the 5- and the 2-day cultures, respectively. Protein expression of these genes can stimulate inflammatory immune responses but also promote tissue repair by attracting curative cells such as endothelial cells (EC) leading to re-establishment of the vasculature. We have established a novel technique by formation of composite islets using EC and mesenchymal stem cells (MSC). EC adhered on the surface of the islets and created a potential blood tolerant surface. The EC-islets showed a degree of protection from the detrimental effects of instant blood-mediated inflammatory reaction (IBMIR) with the major components of IBMIR being decreased in in vitro assays. We combined MSC to the EC-islets with success. The MSC were found to have proliferative effect on EC and the combination of these two cell types on the islets further increased the EC covered surface compared to EC-islets. The EC-MSC-islets in co-culture formed vessel-like structures both into the islets and out to the surrounding matrix. The MSC enhanced the exogenous EC to form vessel-like network in the EC-MSC-islets indicating vascular support by the MSC. The novel strategy and conditions presented herein could alleviate problems related to survival of the islets by promoting revascularization. This would open up a new era in islet transplantation and allow more patients to benefit from this therapy. / Clinical immunology, islet group

A scourge of Type I and Type II diabetes impacts the health of hundreds of millions worldwide. The number and prevalence of diabetics are expected to rise dramatically in the next two decades. Diabetes is defined by chronic hyperglycemia which can result in a number of detrimental and costly metabolic, renal, cardiovascular and neurological disorders. Identification of at risk individuals and effective blood glucose management are critical to improving diabetic outcomes and preventing hyperglycemic complications. Diabetes prevention and treatment is limited by the understanding of islet function and mass in the diabetogenic and diabetic state. The islets of Langerhans are dispersed throughout the pancreas and comprise <2% of the pancreatic mass. The reclusive nature of islet cells presents unique challenges understanding disease development. No agent capable of exclusively targeting pancreatic β-cells within the islet has been discovered and the lack of targeting agent specificity impedes efforts to: quantify β-cell mass and develop novel therapeutics. We propose β-cell targeting can be improved by targeting unique combinations of receptors simultaneously with multivalent ligands. A synthetic multivalent agent composed of two β-cell specific diabetic therapeutics, glucagon-like peptide-1 (GLP-1) and glibenclamide (Glb), targeted against the GLP-1R and the sulfonylurea-1 receptor (SUR1) is a lead compound for the development of specific bi-functional islet cell targeting agents for use in the in vivo detection and treatment of β -cells. Herein, we describe the synthesis and initial characterization of a heterobivalent ligand composed of GLP-1 coupled to Glb. The heterobivalent ligand binds to an unaltered β-cell line with increased specificity relative to a human pancreatic exocrine cell line. Additionally, receptor cross-linking modifies β-cell signaling. Exposure of β-cells to the heterobivalent ligand results in antagonism of SUR1-Ca²⁺ signaling and equipotent agonism of GLP-1R-cAMP signaling, in comparison to the cognate monomeric ligands (Glb and GLP-1). Perturbations in intracellular signaling modifies β-cell insulin secretion resulting in decreased basal insulin secretion and with maintained yet reduced ability to potentiate β-cell glucose stimulated insulin secretion. GLP-1/Glb β-cell specificity and functional modulation suggests combinatorial receptor targeting is an effective strategy for the development of bi-functional cell-specific targeting agents, warranting further investigation and optimization.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

La transplantation d’îlots de Langerhans microencapsulés est un traitement prometteur du diabète de type 1. La microcapsule protège l’îlot du système immunitaire, tout en permettant la diffusion de petites molécules. Comme la microcapsule empêche la revascularisation des îlots, leur oxygénation se fait par diffusion d’oxygène et ils sont exposés à l’hypoxie. Le manque d’oxygène est un facteur limitant dans la survie des îlots microencapsulés. Il est connu que les plus petits îlots sont plus résistant à l’hypoxie à cause d’une meilleure diffusion de l’oxygène. À cette fin, les agrégats de cellules dispersées d’îlots seront étudiés. Lorsque les cellules des îlots sont dispersées, elles ont la propriété de se ré-assembler dans une structure semblable à celle des îlots. La présente étude a permis de mettre au point une technique de formation des agrégats, de les caractériser et de comparer la résistance à l’hypoxie des îlots et des agrégats. Ceux-ci ont une structure semblable aux îlots et ils sont de plus petite taille. Pour cette raison, ils sont plus viables après un choc hypoxique tout en renversant efficacement l’hyperglycémie de souris diabétiques. Les agrégats sont une alternative intéressante pour la transplantation d’îlots microencapsulés puisque leur oxygénation est plus efficace. / Transplantation of microencapsulated islets of Langerhans is a promising treatment for type 1 diabetes mellitus. The microcapsule allows the diffusion of small molecules, while protecting the islet from the antibodies and immune cells. However, microcapsule prevents islet revascularization, thus oxygenation depends on diffusion and islets are exposed to hypoxia. Poor oxygenation is a major limitation in microencapsulated islet survival. It was shown that smaller islets are more resistant to hypoxia because of a better oxygen diffusion. In this study, dispersed islet cell aggregates will be used to improve the oxygenation. When islet cells are dispersed into single cells, they have the ability to re-associate into an islet-like structure. This study allowed to set up a technique to form aggregates, to characterized them and to compare the resistance to hypoxia of islets and aggregates. Aggregates have a similar structure than islets and they are smaller. For this reason, they survive better to a hypoxic treatment, while restoring efficiently normoglycemia in diabetic mices. Aggregates are an interesting solution for microencapsulated islet transplantation because they have a better oxygenation.