O tamanduá-bandeira Myrmecophaga tridactyla é uma espécie considerada “vulnerável” no Brasil, por estar ameaçado de extinção em algumas regiões do país. O presente projeto teve por objetivo identificar e caracterizar as estruturas macro e microscópicas do pâncreas nessa espécie. Para tanto, foram dissecados 16 pâncreas de tamanduás-bandeira provenientes do Hospital Veterinário “Dr. Halim Atique” do Centro Universitário de Rio Preto (UNIRP). As amostras coletadas, foram provenientes de casos de animais atendidos no referido Hospital e que vieram a óbito. O pâncreas situava-se no antímero esquerdo do corpo do animal, apresentava coloração pálida, corpo central e superfície lobulada. Acompanhava a curvatura ventricular maior do estomago aderindo-se na porção inicial do duodeno. Relaciona-se crâniodorsalmente com o baço e ventrículo gástrico, e caudoventralmente com a cápsula fibrosa renal (que aloja o rim esquerdo) e intestinos. Estruturalmente, o órgão demonstrou duas partes distintas: a primeira delas com características exócrinas, composta por ácinos pancreáticos e a segunda endócrina, formada pelas ilhotas pancreáticas encontradas nas regiões media, caudoventral e lobar esquerda. A analise ultraestrutural permitiu identificar nas células centro-acinosas do pâncreas vesículas com grânulos de zimogênio, mitocôndrias, Aparelho de Golgi e retículo endoplasmático rugoso / The giant anteater Myrmecophaga tridactyla is a species considered 'vulnerable' in Brazil since it is threatened in some Brazilian regions. This study aimed to identify and characterize morphological structures of the pancreas in this species. For this, 16 anteaters pancreas from the Veterinary Hospital "Dr. Halim Atique at University Center of Rio Preto (UNIRP), were dissected. All samples were from animals treated at the hospital which died of natural causes. The pancreas was located in the left antimere of the animal’s body, being lobulated and having a pale color and central body. It followed the greater curvature of the stomach, adhering on the initial portion of the duodenum. It was craniodorsally related to the spleen and gizzard, and caudoventrally to the renal fibrous capsule (which houses the left kidney) and intestines. Structurally, the organ had two distinct parts: an exocrine, composed of pancreatic acini; and and endocrine, formed by pancreatic islets found in the medial, caudoventral and left lobar regions. The ultrastructural analysis allowed identifying the central-acinar pancreatic cells with vesicles zymogen granules, mitochondria, Golgi apparatus and rough endoplasmic reticulum

Hintergrund/Ziel: Um Langzeitkomplikationen bei Diabetes mellitus Typ 1 (T1D) zu verhindern, können Spenderinseln transplantiert werden, was allerdings lebenslange rigorose Immunsuppression erfordert. Durch Verkapselung der Inselzellen kann die Immunsuppression umgangen werden, aber Upscaling ist schwierig. Ziel dieser Arbeit war es, eine Strategie für das Plotting skalierbarer, semipermeabler, makroporöser Scaffolds mit einem großen Oberfläche-zu-Volumen-Verhältnis zu entwickeln, um lebensfähige und funktionsfähige Pankreasinseln zu Plotten. Methoden: Die für diese Arbeit verwendete Hydrogelmischung bestand aus 3 % Alginat mit 9 % Methylcellulose (MC) als Verdickungsmittel, um eine plottbare Paste herzustellen. Die existierende Mischung musste für die Verwendung von hochaufgereinigtem, d. h. nicht immunogenem ('clinical-grade') Alginat anstelle des zuvor verwendeten zellverträglichen, aber weniger streng aufgereinigten ('research-grade') Alginates adaptiert werden. Für die Charakterisierung der zellfreien Mischung wurden immer Vergleiche zwischen Pasten gezogen, die mit den beiden verschiedenen Alginaten hergestellt wurden. Die Stabilität wurde über rheologische Messungen und die Freisetzung von Ionen und MC bestimmt. Die Permeabilität für Glukose und Insulin wurde mittels Aufnahme- und Freisetzungs-Assays sowie in einem für diese Arbeit entwickelten Diffusionskammersystem analysiert. Da sich Alginatgele als ausreichend permeabel erwiesen haben und eingekapselte Inseln ihre Funktionalität behalten, wurde die Permeabilität von Alginat- und Alg/MC-Gelen verglichen. In früheren Veröffentlichung wurde Alg/MC bereits als kompatibel mit dem Plotting von Einzelzellen beschrieben. Um die Kompatibilität mit endokrinen Zellen zu testen, wurde die β Zelllinie INS-1 verwendet und in Alg/MC Pasten, die mit unterschiedlich sterilisierter MC hergestellt worden waren, getestet. Für pankreatische Inseln, die als Zellcluster empfindlicher auf Scherstress reagieren als Einzelzellen, wurde erfolgreich ein Workflow für das Einbringen in die hochviskose Mischung entwickelt. Dabei wurden die Inseln mit einem Spatel vorsichtig in das Material gefaltet und mit einer Nadel mit einem Innendurchmesser von 840 µm geplottet. Das Plotten pankreatischer Inseln wurde sowohl mit adulten Inseln aus der Ratte als auch mit neonatalen insel-ähnlichen Clustern aus dem Schwein (NICC) durchgeführt und auf Verteilung, Überleben, Apoptose und das Vorhandensein von Hormonen mit Färbungen getestet. Die Funktionalität wurde über Glukose-stimulierter Insulinsekretion (GSIR) analysiert, wobei Inseln Insulin in Reaktion auf hypoglykämische (3,3 mM Glukose) oder hyperglykämische (16,4 mM Glukose) Bedingungen freisetzen, welches im Überstand nachgewiesen wurde. Ergebnisse: Pasten, die mit den verschiedenen Alginaten hergestellt wurden, zeigten eine vergleichbare Viskosität, die für die Herstellung stabiler Strukturen geeignet ist. Clinical-grade Scaffolds hatten eine leicht geringere Vernetzungsdichte, was auf Unterschiede im M:G-Verhältnis zurückgeführt wurde. Mit 70 mM SrCl2 vernetzte Scaffolds blieben in RPMI+ über 21 Tage stabil. Die anhaltende Freisetzung von Vernetzungsionen über den Kultivierungszeitraum war unabhängig vom Alginattyp, aber abhängig von der Art des verwendeten Mediums. Innerhalb der ionisch vernetzten Scaffolds liegt die umgekehrt thermisch gelierende MC bei 37°C mit hoher Wahrscheinlichkeit teilweise geliert vor. Die Freisetzung von nicht gelierter MC konnte in Abhängigkeit der Temperatur gezeigt werden und wurde in allen getesteten Scaffold-varianten in unterschiedlicher Menge beobachtet. Bei Permeabilitätsanalysen folgte die im Kammersystem beobachtete allgemeine Kinetik der Diffusion dem normalen Verlauf der Diffusion durch Hydrogele. Die Permeabilität für Glukose war zwischen den Materialien vergleichbar, d.h. es konnte weder ein Einfluss des Alginat-Typs und der Vernetzungsdichte, noch des Vorhandenseins bzw. der Freisetzung von MC über die Zeit nachgewiesen werden. Die Permeabilität für Insulin muss aufgrund der Bindung an die Diffusionskammer und der mangelnden Stabilität der Gelscheiben in weiteren Experimenten verifiziert werden. Eine vorläufige Schlussfolgerung aus den hier vorgestellten Ergebnissen ist eine leicht verringerte Permeabilität in Alg/MC-Gelen im Vergleich zu reinen Alginatgelen. INS-1 wurden erfolgreich in Alg/MC geplottet. Die Überlebensrate direkt nach dem Plotten war in Anbetracht der Raten immortalisierter mesenchymaler Stammzellen vergleichsweise gering, INS-1 erholten sich jedoch innerhalb einer Woche und proliferierten innerhalb der Scaffolds zu großen metabolisch aktiven Zellclustern. Dies war abhängig von der Sterilisationsmethode: Die Verwendung autoklavierter sowie UV-sterilisierter MC resultierte in Pasten die das Überleben unterstützten. Im Gegensatz dazu führte die Verwendung von mit überkritischem CO2 sterilisierter MC nicht zur Entwicklung von Zellclustern. Metabolisch aktive, Insulin enthaltende Ratteninseln lagen innerhalb der Scaffolds gleichmäßig verteilt vor. Vergleichbar mit Kontrollinseln in Suspensionskultur betrug die Viabilität geplotteter Inseln über einen Zeitraum von 14 Tagen 70-80 %. Der DNA-Gehalt der Scaffolds reduzierte sich über insgesamt 21 Tage stark. In allen über 7 Tage analysierten Inseln war eine begrenzte Menge apoptotischer Kerne vorhanden. In den Kontrollinseln waren diese bevorzugt zentral, in den geplotteten Inseln bevorzugt peripher lokalisiert. Die Anzahl der apoptotischen Kerne unterschied sich zwischen den Kontrollinseln und den geplotteten Inseln nicht signifikant. Insgesamt blieben die Morphologie und die Viabilität der Inseln während des Einbringens in die Mischung und beim Plotten erhalten. Die pankreatischen Hormone Insulin und Glukagon wurden in der Kontrolle und der geplotteten Inseln in angemessener Verteilung und Lokalisation nachgewiesen. Die Stimulation der Ratteninseln wurde über insgesamt 7 Isolationen verifiziert und zeigte eine geringere ab-solute Insulinsekretion aus den geplotteten Inseln als aus den Kontrollinseln, aber an Tag 4 und 7 der Kultur einen vergleichbaren aus dem GSIR berechneten Stimulationsindex (SI). Darüber hinaus konnte gezeigt werden, dass die Insulinsekretion bei sukzessiv variierender Stimulation mit Glukose dem Verlauf der Glukosekonzentration folgt. Dies zeigt, dass die geplotteten Inseln die externe Glukosekonzentration nachweislich wahrnehmen und entsprechend reagieren. Zusammenfassend lässt sich festhalten, dass die absolute Insulinsekretion aus den geplotteten Inseln zwar geringer war als die der Kontrollinseln, die relative Funktionalität in den geplotteten Scaffolds jedoch erhalten blieb. Zur Validierung der Ergebnisse von adulten Ratteninseln wurden in einer vorläufigen Studie die potentiell klinisch translatierbaren, aber empfindlicheren, NICC verwendet. Metabolisch aktive, Insulin enthaltende NICC, lagen innerhalb der Scaffolds gleichmäßig verteilt vor. Die Viabilität der NICC war mit etwa 60 % vergleichsweise gering, aber vergleichbar mit der der Kontroll-NICC und änderte sich über einen Kultivierungszeitraum von 21 Tagen nicht signifikant. In allen geplotteten NICC konnten lebendige Zellen nachgewiesen werden, während eine geringe Anzahl Kontroll-NICC in der Lebendfärbung fast kein Signal zeigten. Die Anzahl der apoptotischen Kerne nahm im Verlauf von 7 Tagen bei den geplotteten, nicht aber bei den Kontroll-NICC signifikant zu. Die pankreatischen Hormone Insulin, Glukagon und Somatostatin wurden in einer geringen Anzahl von zufällig innerhalb der Cluster verteilten Zellen über einen Zeitraum von 7 Tagen nachgewiesen. Die Funktionalität wurde weder durch das Einbringen in das Material noch durch das Plotten beeinflusst. Unter Verwendung eines GLP-1-Analogons zur Amplifikation der Reaktion zeigten geplottete und Kontroll-NICC eine vergleichbare Funktionalität. Der SI nahm im Laufe der Kultivierungszeit jedoch ab und lag nach 14 bzw. 21 Tagen Kultur in geplotteten und Kontrollproben unter 2. Schlussfolgerungen: Die adaptierte Alg/MC-Mischung zeigte ausreichende Stabilität und Permeabilität für das Plotten pankreatischer Inseln. In einer Proof-of-Concept-Studie mit adulten Ratteninseln wurde gezeigt, dass die hier adaptierte Alg/MC-Mischung generell für das Plotting lebendiger und funktionaler pankreatischer Inseln geeignet ist. Vorläufige Ergebnisse, die mit einer geringen Anzahl von Wiederholungen und Proben erstellt wurden, deuten darauf hin, dass Alg/MC auch ein grundlegend geeignetes Material für das Plotten von NICC ist. Die weitere Charakterisierung und insbesondere die weitere Materialadaption zur Unterstützung des Überlebens und der Ausreifung von NICC in vitro werden in einer Folgestudie vorgenommen werden.:Table of contents Abbreviations 1 1 Motivation 3 2 Introduction and state of the art 5 2.1 Pancreatic islets and diabetes mellitus type 1 5 2.1.1 Anatomy and function of the pancreas and pancreatic islets 5 2.1.2 Insulin biosynthesis, release and function 7 2.1.3 Diabetes mellitus type 1 (T1D) 9 2.1.4 Current treatment options for T1D 11 2.2 Surgical replacement of β-cells 13 2.2.1 Islet transplantation 13 2.2.2 Xenotransplantation 15 2.2.3 Encapsulation of islets 17 2.3 3D bioprinting in medical research 22 2.3.1 Extrusion-based 3D bioprinting 22 2.3.2 Bioplotting of islets 24 3 Materials & Methods 26 3.1 Cell culture 26 3.1.1 Cell lines 26 3.1.2 Primary islets from rat 26 3.1.3 Neonatal porcine islet-like cell clusters 27 3.2 Material preparation and characterisation 27 3.2.1 Hydrogel preparation 27 3.2.2 3D plotting of cell-free hydrogels for material characterisation 28 3.2.3 Rheological characterisation 28 3.2.4 Quantification of ion release 29 3.2.5 Determination of methylcellulose content 29 3.2.6 Preparation of cell-free hydrogel discs 29 3.2.7 Permeability measurements 30 3.3 Plotting and characterisation of cell-laden constructs 32 3.3.1 Incorporation of cells & scaffold preparation 32 3.3.2 Staining methods for the characterisation of (embedded) cells 32 3.3.3 Functional analysis of islets: Glucose stimulated insulin release (GSIR) 34 3.4 Statistics 35 4 Results 36 4.1 Adaptation & characterisation of cell-free Alg/MC 36 4.1.1 Paste viscosity and scaffold stability 36 4.1.2 Scaffold composition during incubation under cell culture conditions 38 4.1.3 Permeability for glucose & insulin 42 4.2 Adaptation & characterisation of islet cell incorporation into the Alg/MC blend 58 4.2.1 Sterilisation of MC: influence on β-cell survival and behaviour 58 4.2.2 Incorporation of pancreatic islets into the highly viscous Alg/MC blend 61 4.2.3 Needle diameter for the plotting of pancreatic islets 64 4.3 Plotting of adult murine pancreatic islets 66 4.3.1 Distribution, morphology and viability of bioplotted murine islets 66 4.3.2 Functionality of bioplotted murine islets 74 4.4 Plotting of neonatal porcine islet-like cell clusters (NICC) 80 4.4.1 Distribution, morphology and viability of bioplotted NICC 80 4.4.2 Functionality of bioplotted NICC 87 5 Discussion 92 5.1 Cell-free Alg/MC 93 5.1.1 Gel viscosity and crosslinking of alginate 93 5.1.2 Release of MC 99 5.1.3 Permeability for glucose & insulin 106 5.2 Cell incorporation into Alg/MC 118 5.2.1 Incorporation of β-cells 118 5.2.2 Incorporation of pancreatic islets 119 5.3 Plotting of adult murine pancreatic islets 121 5.3.1 Distribution, morphology and viability of bioplotted murine islets 121 5.3.2 Functionality of bioplotted murine islets 124 5.4 Plotting of neonatal porcine islet-like cell clusters (NICC) 127 5.4.1 Distribution, morphology and viability of bioplotted murine islets 127 5.4.2 Functionality of bioplotted NICC 130 Summary 134 Zusammenfassung 137 References 140 Addendum 172 A.1 Supplementary data for the results 172 Supplementary data for “4.1.1. Paste viscosity and scaffold stability” 172 Supplementary data for “4.1.2 Scaffold composition during incubation under cell culture conditions” 173 Supplementary data for “4.1.3 Permeability for glucose & insulin” 175 Supplementary data for “4.2.1 Sterilisation of MC: influence on β-cell survival and beha-viour” 178 Supplementary data for “4.3.2 Functionality of bioplotted murine islets” 182 Supplementary data for “4.4.2 Functionality of bioplotted NICC” 187 A.2 Supplementary data for the discussion 189 Supplementary data for “5.1.1. Gel viscosity and crosslinking of alginate” 189 Supplementary data for “5.1.2. MC release” 191 Supplementary data for “5.1.3 Permeability for glucose & insulin” 195 Supplementary data for “5.2.1 Incorporation of β-cells” 197 Supplementary data for “5.4.1 Plotting of neonatal porcine islet-like cell clusters (NICC)” 198 List of figures 196 List of tables 197 List of publications 198 Acknowledgements 200

No description available.

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

As espécies reativas de oxigênio são capazes de influenciar a secreção de insulina, porém ainda não está clara a influência da glicose, principal secretagogo deste hormônio, sobre a homeostase redox das células beta pancreáticas. Incubações por 1 e 48 horas com diferentes concentrações de glicose (2,8; 5,6; 8,3; 11,1; 16,7 e 20 mM) demonstraram que esta é capaz de alterar não só o conteúdo de superóxido, produzido pela mitocôndria e NADPH oxidase, mas também o sistema antioxidante, alterando a concentração de GSH e a expressão das enzimas antioxidantes. Além disso, aumenta a interação Rac1/Sod1, que mantém a NADPH oxidase ativa. Porém, não apresenta endossomas de sinalização redox, os redoxossomas, em resposta a glicose. Estas alterações podem afetar eventos chave para este tecido endócrino, como a secreção de insulina e a morte celular. / ROS production in pancreatic beta cells has been associated with the insulin secretion process but the mechanism by which glucose affects the redox state in these cells remains unknown. In order to address this issue, we evaluated the effect of 1 or 48 hours incubation of pancreatic beta cells with various glucose concentrations (2.8, 5.6, 8.3, 11.1, 16.7 and 20 mM). Glucose loading induced superoxide production by mitochondria and NADPH oxidase complex, and enhanced the antioxidant capacity by increasing GSH content and modulate expression of antioxidant enzymes. Glucose also promoted Rac1/Sod1 interaction that maintains NADPH oxidase activated. These cells however did not present redox endosomes, the redoxosomes, in response to glucose loading. These effects might be associated with the process of insulin secretion and pancreatic beta cell death.

O Diabetes Mellitus tipo 2 (DM 2) é uma doença crônica na qual os pacientes apresentam capacidade secretória de insulina inadequada para suplantar a resistência insulínica concomitante e, como resultado, advém a hiperglicemia. Os mecanismos que explicam a diminuição da secreção insulínica não são completamente conhecidos e acredita-se que o depósito de amilina, um achado histopatológico freqüente nesses pacientes, esteja envolvido. A amilina humana é uma proteína co-secretada com a insulina capaz de se agregar e se depositar nas ilhotas pancreáticas. Ainda não está totalmente estabelecido se a toxicidade da amilina humana é mediada pelas fibrilas maduras, conforme demonstrado em trabalhos mais antigos, ou por oligômeros de tamanho intermediário, como tem sido aventado nos trabalhos mais recentes. O objetivo deste estudo foi avaliar o perfil de genes modulados por oligômeros, bem como por fibrilas maduras de amilina, em ilhotas pancreáticas de rato. As ilhotas foram isoladas a partir de ratos Wistar, mantidas em cultura por 24 horas e a seguir tratadas com 10 M de oligômeros ou de fibrilas maduras de amilina por 24 horas adicionais em concentração fisiológica ou suprafisiológica de glicose. O RNA total foi extraído e utilizado para análise da expressão gênica por microarranjos de DNA. O conteúdo de RNA de alguns genes modulados nas condições experimentais estudadas também foi avaliado por RT-qPCR, a fim de validar os resultados obtidos pela análise de microarranjos. A análise das vias significativamente afetadas pelas preparações de amilina demonstrou que, em ilhotas mantidas em concentração fisiológica de glicose, os oligômeros de amilina modularam, entre outros, processos relacionados à Resposta ao Estresse e à Apoptose, processos não modulados pelas fibrilas maduras de amilina. Em concentração suprafisiológica de glicose, o tratamento com oligômeros de amilina deixou de modular as vias relacionadas a Estresse e Apoptose, surgindo como moduladas vias relacionadas aos processos de Regulação da endocitose e Biossíntese de óxido nítrico. Os resultados do RT-qPCR sugeriram que somente os oligômeros (e não as fibrilas maduras) de amilina modulam genes relacionados a apoptose (Anxa1, Rab5a) e ao estresse oxidativo (Nos2 e Xdh), o que vai ao encontro dos estudos mais recentes que atribuem às fibrilas intermediárias um papel na citotoxicidade das células . Um achado novo do presente estudo foi a identificação do mRNA do Gipr (receptor de polipeptídeo inibitório gástrico) como alvo de regulação negativa pelos oligômeros de amilina, o que sugere que esse possa ser um mecanismo adicional pelo qual essas fibrilas intermediárias de amilina sejam deletérias para a célula pancreática. / Type 2 diabetes mellitus is a chronic disease in which there is inability of pancreatic cells to secrete sufficient insulin to overcome the insulin resistance in the peripheral tissues with resultant hyperglycemia. Mechanisms leading to diminished insulin secretion are not completely known and the amyloid deposit, a frequent histopathological finding in patients with type 2 diabetes, is believed to be involved. Human amylin, a protein co-secreted with insulin, is capable of aggregating and forming deposits in the pancreatic islets. It is not fully established whether amylin cytotoxicity is mediated by mature amylin fibrils or by soluble oligomers. The objective of this study was to evaluate the gene profiling modulated by oligomers as well as by mature amylin fibrils in rat pancreatic islets. The islets were isolated from Wistar rats, maintained in culture for 24 hours and then treated with 10 M of oligomers or mature amylin fibrils for additional 24 hour in physiologic and supraphysiologic glucose concentrations. Total RNA was extracted and used for gene expression analysis by microarray. RNA content of some modulated genes was evaluated by RT-qPCR in order to validate the results obtained from the microarray analysis. The analysis of the pathways significantly affected by the two amylin preparations demonstrated that, in islets maintained in physiological glucose concentration, amylin oligomers modulated, among others, processes related to Response to stress and to Apoptosis, which were not modulated by mature amylin fibrils. In supraphysiological glucose concentration, treatment with oligomers did not modulate the pathways related to Stress and Apoptosis, which were replaced by processes related to Endocytosis regulation and Nitric oxide biosynthesis. RT-qPCR results suggested that only amylin oligomers modulate genes related to apoptosis (Anxa1, Rab5a) and oxidative stress (Nos2 e Xdh), which is in agreement with studies indicating a role for oligomers in the cytotoxicity of cells. A new finding of the present study was the identification of the Gipr (gastric inhibitory polypeptide receptor) mRNA as a target for downregulation by amylin oligomers, which suggests that this might be an additional mechanism by which these oligomers are deleterious to the pancreatic cells.

<p>The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied <i>in vitro</i> in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound.</p><p>It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which <i>in vivo</i> may increase a virus’s ability to persist in islets.</p><p>Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect.</p><p>All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis <i>in vivo</i>.</p><p>Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study.</p><p>To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells <i>in vivo</i>, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction <i>in vivo</i>.</p>

<p>Type 1 diabetes mellitus affects millions of people worldwide. Islet transplantation is a minimal invasive surgical procedure that restores euglycemia and halts the progression of diabetic complications. However, despite transplantation of islets from multiple donors most patients reverse to hyperglycemia within five years. New strategies to improve long-term outcome of islet transplantation are indispensable. This thesis studied differences in the microvasculature between endogenous and transplanted pancreatic islets, and investigated means to improve islet graft revascularization and function. Islet graft microvessels were similar to endogenous islets responsive to adenosine, angiotensin II and nitric oxide (NO). Recipient hyperglycemia induced a higher basal islet graft blood flow, which also was less dependent on NO than in normoglycemic recipients. Transplantation of freshly isolated instead of cultured islets improved graft revascularization, oxygenation and function. Pretreatment of islets with vascular endothelial growth factor decreased their expression of matrix metalloproteinase-9 (MMP-9) and impaired graft revascularization. Moreover, MMP-9 pretreatment <i>per se</i> improved graft revascularization. <i>In vivo</i>, 20-25% of all endogenous rat islets was low oxygenated (pO₂ <10 mmHg). Changes in the islet mass, by means of whole-pancreas transplantation, doubled the fraction of low oxygenated islets in the endogenous pancreas of transplanted animals, whereas this fraction almost completely disappeared after a 60% partial pancreatectomy. Interestingly, oxygenation was related to metabolism, since well oxygenated islets <i>in vivo</i> had 50% higher leucine-dependent protein biosynthesis, which includes (pro)insulin biosynthesis. In intraportally transplanted islets, the low oxygenated fraction of islets was markedly increased one day post-transplantation, and the oxygenation remained low following revascularization. In summary, these data suggest that a better revascularization of transplanted islets can improve graft function. Furthermore, the oxygenation and metabolism of endogenous islets is tightly regulated. This regulation seems to be disturbed following transplantation, which may contribute to long-term islet graft failure. </p>

The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied in vitro in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound. It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which in vivo may increase a virus’s ability to persist in islets. Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect. All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis in vivo. Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study. To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells in vivo, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction in vivo.

Type 1 diabetes mellitus affects millions of people worldwide. Islet transplantation is a minimal invasive surgical procedure that restores euglycemia and halts the progression of diabetic complications. However, despite transplantation of islets from multiple donors most patients reverse to hyperglycemia within five years. New strategies to improve long-term outcome of islet transplantation are indispensable. This thesis studied differences in the microvasculature between endogenous and transplanted pancreatic islets, and investigated means to improve islet graft revascularization and function. Islet graft microvessels were similar to endogenous islets responsive to adenosine, angiotensin II and nitric oxide (NO). Recipient hyperglycemia induced a higher basal islet graft blood flow, which also was less dependent on NO than in normoglycemic recipients. Transplantation of freshly isolated instead of cultured islets improved graft revascularization, oxygenation and function. Pretreatment of islets with vascular endothelial growth factor decreased their expression of matrix metalloproteinase-9 (MMP-9) and impaired graft revascularization. Moreover, MMP-9 pretreatment per se improved graft revascularization. In vivo, 20-25% of all endogenous rat islets was low oxygenated (pO2 &lt;10 mmHg). Changes in the islet mass, by means of whole-pancreas transplantation, doubled the fraction of low oxygenated islets in the endogenous pancreas of transplanted animals, whereas this fraction almost completely disappeared after a 60% partial pancreatectomy. Interestingly, oxygenation was related to metabolism, since well oxygenated islets in vivo had 50% higher leucine-dependent protein biosynthesis, which includes (pro)insulin biosynthesis. In intraportally transplanted islets, the low oxygenated fraction of islets was markedly increased one day post-transplantation, and the oxygenation remained low following revascularization. In summary, these data suggest that a better revascularization of transplanted islets can improve graft function. Furthermore, the oxygenation and metabolism of endogenous islets is tightly regulated. This regulation seems to be disturbed following transplantation, which may contribute to long-term islet graft failure.