Prevalence of type 2 diabetes mellitus (T2DM) is still rising and even so in the juvenile population. Obesity is highly associated with increased risk for developing T2DM. The development has been related to elevated fasting concentrations of the pancreatic islet hormones insulin and glucagon as well as to an increase in plasma lipids that occurs during obesity. Specifically, research has indicated that chronic exposure to high levels of saturated free fatty acids cause dysfunction in islet alpha- and beta-cells. Fatty acids can affect islet cells by various mechanisms one of which is the G-protein coupled receptor FFAR1/GPR40. The role of the receptor in the effects of fatty acids on pancreatic islet-cell function is not clear. The aim of this thesis was to clarify the role of FFAR1 in how fatty acids, and more specifically the long-chain saturated fatty acid palmitate, affect insulin and glucagon secretion. In children and adolescents with obesity elevated fasting levels of insulin and glucagon were positively correlated with lipid parameters. Specifically, plasma triglycerides and free fatty acids were positively correlated with insulin and glucagon at fasting as well as with visceral adipose tissue volume. Elevated glucagon levels at fasting were associated with worsening of glucose tolerance in the same population. In in vitro studies of isolated human islets palmitate stimulated basal insulin and glucagon secretion as well as mitochondrial respiration at fasting glucose levels. The effect was mediated by FFAR1 and fatty acid beta-oxidation. At higher glucose concentrations the receptor was involved in the potentiation of insulin secretion from isolated human islets and insulin-secreting MIN6 cells. Furthermore, we found that the effects of palmitate on hormone secretion were associated with enhanced mitochondrial respiration mediated by FFAR1 Gαq signaling and PKC activity as well as increased intracellular metabolism induced by the fatty acid. When islets were exposed to palmitate for long time periods and in the presence of FFAR1 antagonist, normalized insulin and glucagon secretion during culture and insulin response to glucose after culture were observed. In MIN6 cells chronic palmitate treatment increased mitochondrial uncoupling irrespective of FFAR1 involvement. However, FFAR1 antagonism during palmitate exposure resulted in elevated respiration and reduced apoptosis. In conclusion, children and adolescents with obesity have elevated fasting concentrations of insulin and glucagon that correlate with free fatty acids and fatty acid sources. High glucagon levels are linked to worsening of glucose tolerance in these subjects. In vitro the combination or synergy of FFAR1 activation and intracellular metabolism caused by palmitate is decisive for both the short-term enhancement effects and the negative chronic effects on insulin and glucagon secretion.

Type 1 diabetes (T1D) results from a loss of functional insulin-producing pancreatic beta cells. The etiology of T1D is poorly understood, but the detection of infiltrating inflammatory cells in the pancreas and circulating autoantibodies has led to the common notion that an autoimmune process plays a central role in the pathogenesis of the disease. The aim of this doctoral thesis was to assess various aspects of the immunopathology of type 1 diabetes. To this purpose, studies have been conducted on pancreatic material from the Network for Pancreatic Organ Donors with Diabetes (nPOD) collection, the Nordic Network for Islet Transplantation, and the Diabetes Virus Detection (DiViD) study. Paper I is a study on pancreatic tissue from organ donors with varying duration of T1D as well as non-diabetic donors and subjects with other types of diabetes, in which persistent expression of glucose transporters was shown on the beta cell membrane despite several years of T1D. Glucose transporter 1 was also confirmed as the predominant glucose transporter on human pancreatic islets. In paper II, we report on signs of inflammation in the exocrine but not in the endocrine pancreas in non-diabetic organ donors with diabetes-related autoantibodies, suggesting that diabetes-associated autoantibodies can occur in response to unspecific pancreatic lesions. Paper III aimed to characterize the T cell-infiltration of pancreatic islets in material from recent-onset T1D patients. Insulitis was shown in all subjects, but with distinct differences in expression analysis of T- and B cell activation to cell-mediated allorejected kidney transplant. Also Paper IV was conducted on material from recent-onset cases and showed increased islet glucagon content, in combination with a reduced number of islets but sustained mean islet size. Together, these results provide expansion of our knowledge of the immunopathology in T1D, and will hopefully assist in bringing us towards a deeper understanding of T1D aetiology and eventually an effective cure.

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.

Insulin-produzierende Zellen als Ersatz für die beim Diabetes mellitus Typ 1 zerstörten Betazellen stellen einen hochattraktiven Forschungsansatz dar. Ziel dieser Arbeit war, Insulin-positive Zellen aus in vitro modifizierten Blutmonozyten zu gewinnen. Blutmonozyten sind nicht nur, wie bereits seit längerem bekannt, in der Lage, sich in Makrophagen und dendritischen Zellen zu differenzieren, sondern auch in eine Vielzahl nicht-phagozytierender Zellen, wie z.B. Insulin-produzierender Zellen. Für die optimale Zelltherapie ist zu fordern, dass die gewünschten Zellen in vivo nicht nur ihre Funktion beibehalten, sondern dass von diesen Zellen auch kein immunologisches Risiko für den Patienten ausgeht. Eine dauerhafte Immunsuppression, wie sie für die Vollorgantransplantation notwendig ist, ist für Zelltransplantate nicht angebracht. Hier besteht Übereinkunft, dass Immunsuppressiva, wenn überhaupt, nur kurzfristig einzusetzen sind. Blutmonozyten lassen sich einfach gewinnen und stünden somit als autologer Zellersatz für eine mögliche Zelltherapie zur Verfügung. Ein wesentlicher Aspekt dieser Arbeit war, die in vitro Differenzierung von Blutmonozyten zu charakterisieren. Dabei sollte die Expression von Insulin, Gluka¬gon und dem Glukosetransporter Glut-2 nachgewiesen werden. Auch morpho¬logische Veränderungen während der Kultur sollten beobachtet werden. Die kultivierten Monozyten entwickelten sich mit zunehmender Kulturdauer eindeutig zu Makrophagen. Dabei waren zwei verschiedene Zellmorphologien zu unterscheiden: Der erste Zelltyp (Typ 1) war oval mit Ausläufern. Der zweite Zelltyp (Typ 2) war sehr groß, teilweise mit einem Durchmesser von über 500 μm, häufig von ovaler Form und polynukleär. Dieser Zelltyp wies zudem häufig einen breiten, um das Kerngebiet gruppierten Saum auf. Mit zunehmender Kulturdauer dominierte dieser Zelltyp die Kultur. Der Großteil der Typ 1-Zellen blieb CD14 positiv. Gab es CD14-negative Zellen in der Kultur, so gehörten sie mit großer Wahrscheinlichkeit zu den Typ 2-Zellen. Nur in den in vitro modifizierten, nicht aber in den frisch isolierten Monozyten waren Insulin, C-Peptid, Glukagon und GLUT-2 immunhistochemisch nachzu¬weisen. Mit zunehmender Kulturdauer dominierten stark adhärente Makrophagen die Kultur. Das aus ca. 5x106 Monozyten isolierte Insulin senkte den Blutzuckerspiegel diabetischer Mäuse innerhalb einer Stunde nach Injektion um 66,1±12,8 Prozent (n=5). Zum Vergleich: 170 pg Humaninsulin senkten den Blutzuckerspiegel um 84,2±8,4 Prozent (n=4). Insulin-negative Monozyten beeinflussten nicht den Blutzuckerspiegel diabeticher Mäuse. Zudem lassen erste elektronenmikroskopische Aufnahmen von in vitro modifizierten Monozyten Insulin-haltige Vesikel erkennen. Zum jetzigen Zeitpunkt ist gesichert, dass in vitro modifizierte Monozyten über biologisch aktives Insulin verfügen, das den Blutzuckerspiegel diabetischer Tiere senkt. Der Nachweis von C-Peptid deutet zudem darauf hin, dass es sich hierbei um de novo Insulin handelt. Dies bedeutet, dass das Insulin-Gen in den in vitro modifizierten Monozyten aktiv ist und sie Insulin mRNA exprimieren, die anschließend in Insulin translatiert wird. Der elektronenmikroskopische Nachweis Insulin-haltiger Granula deutet außerdem darauf hin, dass diese Zellen Insulin speichern können. Inwieweit sie jedoch auch zur Glukose-ab¬hängigen Insulin-Ausschüttung in der Lage sind, ist in weiteren Experimenten zu überprüfen. / Promising cell replacement strategies may restore insulin-secretion in patients with type 1 diabetes. Cells suitable for such a strategy must demonstrate prolonged function in vivo and should not induce immunological responses. A chronic immuno¬suppressive therapy which is requisite for organ grafts is not suitable for cell grafts. Peripheral human blood monocytes easily obtained from patients can be modified in vitro into insulin-positive cells and, therefore may be perfect for autologic cell replacement strategies. The purpose of this study was to characterise cultured monocytes for the presence of insulin and C-peptide as a well-defined indicator for insulin synthesis by immunohistochemistry. In addition, the expression of glucagon and the glucose transporter Glut-2 was proved. During culture, monocytes differentiated into cells with unique morphology. The cell type 1 showed a lengthy-oval shape with branches, like fibroblasts. The cell type 2 was very large, oval in shape and often polynuclear. These cells demonstrated the morphology of long-term cultured macrophages and dominated the culture in the course of time. Cultured monocytes were positive for insulin, C-peptide, glucagon and Glut-2 in contrast to freshly isolated monocytes. Evidence from the electron microscopy indicated that insulin-positive monocytes store their insulin in vesicles. Insulin isolated from 5x106 insulin-positive monocytes was able to reduce blood glucose levels of diabetic mice (> 22 mmol/L) about 66.1±12.8 percent (n=5) within one hour after injection. In comparison, 170 pg of human insulin decreased blood glucose levels of diabetic mice about 84.2±8.4 percent (n=4). Insulin-negative monocytes were unable to reduce blood glucose levels. In this study the possibility of modifying monocytes into insulin-positive cells during culture was confirmed. The detection of C-peptide supports the existence of de novo insulin within these cells which was stored in granula and biologically active. In further studies these cells have to prove whether they are really able to secrete insulin in a dose-dependent manner qualifying them as long-term cell replacements.

Made available in DSpace on 2015-04-14T13:34:39Z (GMT). No. of bitstreams: 1 412115.pdf: 1421285 bytes, checksum: 45019602bb21738c0816090a64e131f0 (MD5) Previous issue date: 2009-02-09 / O transplante de ilhotas surge como uma alternativa para o tratamento do diabetes mellitus tipo 1. Entretanto, durante o processo de isolamento, h? grande perda celular, principalmente na periferia da ilhota pancre?tica. As esp?cies reativas de oxig?nio (ROS) contribuem significativamente neste processo afetando a viabilidade destas c?lulas para transplante. V?rios esfor?os est?o sendo feitos na tentativa de minimizar os danos causados pela libera??o e produ??o de ROS. Apresentando importante papel de citoprote??o e redu??o da forma??o e libera??o de radicais livres, a frutose-1,6-bisfosfato ? um regulador metab?lico que estimula as rea??es catab?licas da glic?lise, contribuindo para melhora da preserva??o do tecido frente ? isquemia em ?rg?os como cora??o, f?gado e rins. Diante destas caracter?sticas, a frutose-1,6-bisfosfato pode apresentar uma possibilidade de uso no isolamento de ilhotas na tentativa de promover prote??o celular. Foi realizado um estudo utilizando FBP durante o isolamento de ilhotas pancre?ticas de camundongos, analisando a contagem e a viabilidade celular, assim como a mensura??o de radicais livres. Nesse trabalho n?o foi encontrado efeito nas diferentes concentra??es de FBP versus controle, tanto na contagem de ilhotas pancre?ticas quanto nas suas viabilidades. Na mensura??o dos radicais livres tamb?m n?o foi observado a a??o citoprotetora nas doses de FBP estudadas.

Zhao Hailu. / "June 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 192-210). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Submitted by Samira Prince (prince@ufpa.br) on 2012-10-16T14:14:53Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ModulacaoImunologicaInvitro.pdf: 477216 bytes, checksum: b32e46d44b06218c4e2ebe58d0f28a6b (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-10-16T18:28:31Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ModulacaoImunologicaInvitro.pdf: 477216 bytes, checksum: b32e46d44b06218c4e2ebe58d0f28a6b (MD5) / Made available in DSpace on 2012-10-16T18:28:31Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ModulacaoImunologicaInvitro.pdf: 477216 bytes, checksum: b32e46d44b06218c4e2ebe58d0f28a6b (MD5) Previous issue date: 2008 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As células de Langerhans (CLs) estão localizadas na epiderme e desempenham um papel chave na indução da resposta imune e da tolerância imunológica. Os macrófagos são células fagocíticas que atuam como primeira linha de defesa do organismo, e que estão envolvidos na formação de granulomas em pacientes com hanseníase. A imunopatogenia da resposta celular nos estados reacionais ainda é pouco estudada, porém, diversas evidências sugerem que as drogas prednisona, talidomida, ciclosporina e amitriptilina, utilizadas no controle das reações hansênicas, exercem seus efeitos pela modulação das funções de diferentes células imunocompetentes. O objetivo do presente estudo foi analisar a ação in vitro das drogas prednisona, talidomida, ciclosporina e amitriptilina sobre a produção de citocinas por CLs e macrófagos de camundongos BALB/c. As CLs foram isoladas, purificadas e cultivadas a partir da epiderme pela técnica de “panning” e os macrófagos foram isolados da cavidade peritoneal de camundongos BALB/c. Após 36 h de tratamento com as drogas, os níveis de TNF-, IL-12 e IL-10 foram medidos por ELISA. Prednisona, talidomida, ciclosporina e amitriptilina inibiram os níveis de TNF- produzidos pelas CLs, em ambas as concentrações, no entanto, não foi detectada alteração significativa na produção de IL-12. A produção de TNF- e de IL-12 por macrófagos peritoneais também foi diminuída após o tratamento, porém os níveis de IL-10 não foram modificados por nenhuma das drogas testadas. Nossos resultados mostram que estas drogas podem modular a resposta imune através da regulação das citocinas pró-inflamatórias TNF- e IL-12 por CLs purificadas da epiderme e por macrófagos peritoneais, indicando que as citocinas constituem importante alvo de drogas usadas no tratamento dos estados reacionais. / Langerhans cells (LCs) are localized in the epidermis and performs a key role in the induction of immune response and immunologic tolerance. Macrophages are phagocytic cells that act as first line of defense of the organism, and they are involved in the granuloma formation in patients with leprosy. The immunopathogeny of the cellular response in the reactional states is yet little studied, however, several evidences suggest that the drugs prednisone, thalidomide, cyclosporine and amitriptyline, used in the control of leprosy reactions, perform their effects by the modulation of different immunocompetent cells functions. The objective of the present study was to analyze in vitro action of prednisone, thalidomide, cyclosporine and amitriptyline on the cytokine production by LCs and macrophages of BALB/c mice. LCs were isolated, purified and cultivated from the epidermis by the panning technique and macrophages were isolated by the peritoneal cavity of BALB/c mice. After 36 h of treatment with the drugs, the levels of TNF-, IL-12 and IL-10 were measured by ELISA. Prednisone, thalidomide, cyclosporine and amitriptyline inhibited TNF- produced by LCs, in both concentrations, however no important alterations in IL-12 production were detected. TNF- and IL-12 production by macrophages was also decreased after treatment, but IL-10 levels were not modified for none of the drugs tested. Our results show that these drugs can modulate the immune response by the regulation of pro-inflammatory cytokines TNF- and IL-12 by purified epidermal LCs and peritoneal macrophages, indicating that they constitute an important target for the drugs used in treatment of leprosy reactional states.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-10-01T14:12:49Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_LeishmaniaAmazonensisInibe.pdf: 993739 bytes, checksum: fc0fe2bebcd57504018c72ec17b3b51a (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-10-07T13:52:49Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_LeishmaniaAmazonensisInibe.pdf: 993739 bytes, checksum: fc0fe2bebcd57504018c72ec17b3b51a (MD5) / Made available in DSpace on 2014-10-07T13:52:49Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_LeishmaniaAmazonensisInibe.pdf: 993739 bytes, checksum: fc0fe2bebcd57504018c72ec17b3b51a (MD5) Previous issue date: 2014 / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / Leishmania amazonensis é um dos principais agentes etiológicos em um amplo espectro de formas clínicas da Leishmaniose Tegumentar Americana. De modo geral, a resistência frente às leishmanioses decorre do desenvolvimento de uma resposta imune celular eficiente, porém muitos estudos têm demonstrado que citocinas específicas ou combinações de citocinas podem ser fatores de resistência ou suscetibilidade à infecção por L. amazonensis. Estudos recentes sugerem a participação das células de Langerhans (LCs) nas resposta anti-Leishmania, porém os mecanismos envolvidos durante esta interação são ainda pouco estudados. Objetivos: Estudar o papel do TNF-α e anti-CD40 nas interações in vitro entre as LCs e L. amazonensis, observando o perfil de citocinas produzidas e a expressão de moléculas de superfície, bem como verificar a capacidade destas células em ativar a produção de IFN-γ e IL-4 por células do linfonodo. Metodologia: As LCs foram isoladas da epiderme de camundongos BALB/c e incubadas com promastigotas de L. amazonensis, TNF-α e/ou anti- CD40. Após 24h, as LCs foram co-cultivadas com células obtidas de linfonodos por 72h. As citocinas IL-6, IL-12, IFN-γ e IL-4 foram dosadas por ensaio imunoenzimático (ELISA) e as moléculas de superfície foram analisadas por citometria de fluxo. Resultados: Os níveis de IL- 6 e IL-12p70 produzidos pela LCs foram significativamente reduzidos após interação com L. amazonensis, mesmo após o tratamento das LCs com TNF-α ou anti-CD40. Em relação às moléculas de superfície, não houve diferença na expressão de CD207 em nenhum dos grupos, porém a presença de L. amazonensis promoveu uma redução significativa na expressão de CD40 nas LCs tratadas com TNF-α ou anti-CD40, e aumentou a expressão de CD86 em todos os grupos. Na presença de L. amazonensis, as células do linfonodo apresentaram uma produção diminuída de IFN-γ e não houve alteração na produção de IL-4. Quando cocultivadas com LCs estimuladas previamente com L. amazonensis, a produção de IFN-γ também foi reduzida, mesmo na presença dos estímulos TNF-α e/ou anti-CD40. Não foram observadas alterações significativas na produção de IL-4 pelas células do linfonodo cocultivadas nas mesmas condições experimentais. Conclusão: L. (L.) amazonensis exerce um efeito imunomodulador sobre a resposta imune mediada por LCs, inibindo a produção de IL-6 e IL-12p70 e expressão de CD40, além de impedir a ativação da produção de IFN-γ por células do linfonodo co-cultivadas com LCs, mesmo após tratamento com TNF-α e anticorpo anti-CD40. / Leishmania amazonensis is one of the agents in a wide spectrum of clinical forms of cutaneous leishmaniasis. In general, the resistance against leishmaniasis depends on the development of an efficient immune response, however many studies have demonstrated that specific cytokines or combinations of cytokines may be factors of resistance or susceptibility to infection by L. amazonensis. Recent studies suggest the involvement of Langerhans cells (LCs) in the anti-Leishmania response, but the mechanisms involved in this interaction are still poorly understood. In this study, we investigated the role of TNF-α and anti-CD40 in L. amazonensis interaction with LCs in vitro, showing de profile of cytokines produced and the expression of surface molecules, besides verifing their abilities to activate the production of IFN-γ e IL-4 by lymph node cells. Methods: Fresh immature LCs, highly purified from BALB/c mouse skin, were incubated with L. amazonensis promastigotes, TNF-α and/or anti- CD40 mAb. After 24 h, LCs were co-cultured with lymph nodes cells of BALB/c mice for additional 72h. Culture supernatants were tested for IL-6, IL-12p70, IFN-γ and IL-4 by ELISA, while surface molecules were analyzed by FACS. Results: The levels of IL-6 and IL- 12p70 produced by LCs were significantly reduced after interaction with L. amazonensis, even after treatment of LCs with TNF-α or anti-CD40. Regarding surface molecules, there was no difference in the expression of CD207 in both groups, but the presence of L. amazonensis promoted a significant reduction in the expression of CD40 on LCs treated with TNF-α or anti-CD40, and increased expression CD86 in all groups. Lymph node cells showed a decreased production of IFN-γ in the presence of L. amazonensis and no change in IL-4. When co-cultured with LCs previously stimulated with L. amazonensis, the production of IFN-γ was also reduced, even in the presence of TNF-α and/or anti-CD40. No significant changes were observed in IL-4 by lymph cells co-cultured under the same experimental conditions. Conclusion: L. (L.) amazonensis exert an immunomodulatory effect on the immune response mediated by LCs by: 1) inhibiting the production of IL-6 and IL-12p70; 2) decreasing CD40 expression and; 3) preventing the activation of IFN-γ production by lymph node cells co-cultured with LCs, even after treatment with TNF-α and anti-CD40 antibody.

<p>During the past decade clinical islet transplantation has become a viable strategy for curing type 1 diabetes. The limited supply of organs, together with the requirement for islets from multiple donors to achieve insulin independence, has greatly limited the application of this approach. </p><p>The islets are infused into the liver via the portal vein, and once exposed to the blood, the grafted tissue has been shown to be damaged by the instant blood-mediated inflammatory reaction (IBMIR), which is characterized by coagulation and complement activation as well as leukocyte infiltration into the islets. Islet revascularization is a subsequent critical step for the long-term function of the transplanted graft, which may partially be impeded by the IBMIR. </p><p>In this thesis, we have explored novel strategies for circumventing the effects of the IBMIR and facilitating islet revascularization.</p><p>Systemic inhibitors of the IBMIR are typically associated with an increased risk of bleeding. We therefore evaluated alternative strategies for modulating the islets prior to transplantation. We demonstrated, using an adenoviral vector, that a high level of expression and secretion of the anticoagulant hirudin could be induced in human islets. An alternative approach to limiting the IBMIR was developed in which anticoagulant macromolecular heparin complexes were conjugated to the islet surface. This technique proved effective in limiting the IBMIR in both an in vitro blood loop model and an allogeneic porcine model of islet transplantation. An increased adhesion of endothelial cells to the heparin-coated islet surface was demonstrated, as was the capacity of the heparin conjugate to bind the angiogenic factors VEGF and FGF; these results have important implications for the revascularization process.</p><p>The outcome of the work in this thesis suggests that modulation of the islet surface is an attractive alternative to systemic therapy as a strategy for preventing the IBMIR. Moreover, the same techniques can be employed to induce revascularization and improve the engraftment of the transplanted islets. Ultimately, improved islet viability and engraftment will make islet transplantation a more effective procedure and increase the number of patients whose diabetes can be cured.</p>

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