Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity. In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation in vivo. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF in vitro efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated in vivo in patients transplanted with isolated islets. There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets in vitro and in vivo, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials.

De ekologiskt betydelsefulla småbiotoperna blir allt färre i det moderna jordbruket på grund av intensifieringen av jordbrukslandskapet som resulterat i ett allt mer fragmenterat landskap. För att kunna ge småbiotoper, såsom åkerholmar, ett optimalt skydd behövs flera studier om organismerna på holmarna, deras diversitet och spridning. I denna undersökning studeras artrikedomen av mossor och lavar på busk- och trädbekädda samt öppna åkerholmar i ett öppet- och ett skogsdominerat landskap. Analysen av artrikedom i studien tyder på att de finns fler arter av mossor och lavar i det öppna landskapet i förhållande till det skogsdominerade landskapet samt på de åkerholmarna som saknade busk- och trädvegetation. Dessa miljöer består av torr och stenig mark och växer igen relativt långsamt, vilket är gynnsamt för vissa mossor och lavar. Det fanns ingen signifikant korrelation mellan diversitet och storleken på åkerholmarna eller mellan diversitet och holmarnas avstånd till skog. Antalet arter i ett habitat kan bero på regionala artpoolen samt antalet potentiella kolonisatörer men även lokala processer. Vidare är det viktigt att nämna att studien tyder på att diversitet av mossor och lavar påverkas av förhållandena på holmarna, såsom busk- och trädtäckningen. Detta i kombination med en variation inom dessa små biotoper kan skapa goda förutsättningar för ett landskap med hög diversitet. / The ecologically important small remnant habitats are becoming fewer in modern agriculture due to the intensification of agricultural landscapes that have resulted in an increasingly fragmented landscape. In order to provide an ideal conservation framework for small habitats, such as midfield islets, several studies about the organisms on the islets, their diversity and distribution are needed. In this study I investigated the diversity of mosses and lichens on open midfield islets with and without shrubs and trees in an open and a forest-dominated landscape. The analysis of species diversity in the study suggests that, overall; there are more species of mosses and lichens in the open landscape in relation to the forest-dominated landscape, and on the opened islets lacking shrub and tree vegetation. These environments consist of dry and rocky soils and overgrow relatively slowly, which is favourable for mosses and lichens. There was no significant correlation between diversity and size of the midfield islets or their distance to forest. The number of species in a habitat may depend on regional species pool and the number of potential colonizers but also local processes. Furthermore, it is important to mention that the study suggests that the diversity of mosses and lichens is influenced by the conditions on the midfield islands, such as shrub and tree cover. These conditions, combined with a variation within these small habitats can create good conditions for a landscape with high diversity.

No description available.

Clinical islet transplantation is today an established alternative treatment for a selected group of type 1 diabetes patients. The predominant technique for transplantation is infusion of islets in the liver via the portal vein. Obstacles to advancing islet transplantation include limited engraftment resulting from an immediate blood-mediated inflammatory reaction (IBMIR), a life-long need for immunosuppression and the shortage of organs available. In this thesis, innate and adaptive immunity were explored in allogeneic and xenogeneic settings, with the long-term goal of preventing islet graft destruction. Methods for studying immune responses to islets in blood and engrafted islets in liver tissue (intragraft gene expression) were developed and refined. The innate response to human islets and exocrine tissue in ABO-compatible blood was characterized up to 48 h using a novel whole-blood model. Physiological changes in the blood during incubations were explored and adjusted to allow prolonged experiments. Increased production of chemokines targeting CXCR1/2, CCR2 and CXCR3 was observed, accompanied by massive intra-islet neutrophil infiltration. Notably, endocrine and exocrine tissue triggered a similarly strong innate immune response. Two studies of adult porcine islet transplantation to non-human primates (NHPs) were performed. Expression of immune response genes induced in liver tissue of non-immunosuppressed NHPs (≤72 h) was evaluated after porcine islet transplantation. Up-regulation of CXCR3 mRNA, together with IP-10, Mig, MIP-1α, RANTES, MCP-1 and cytotoxic effector molecule transcripts, was associated with T-cell and macrophage infiltration at 48-72 h. Long-term survival (>100 days) of adult porcine islets in a NHP model was later demonstrated using T-cell-based immunosuppression, including co-stimulatory blockade (anti-CD154 mAb). Graft failure was associated with increased levels of circulating, indirectly activated T cells, non-Gal pig-specific IgG and gene transcripts of inflammatory cytokines. Microarray analysis of the response to inflammatory cytokines in cultured porcine islets identified genes involved in cell death, immune responses and oxidative stress; this gene pattern coincided with physiological changes (decrease in insulin and ATP content). In summary, allogeneic whole-blood experiments and xenogeneic in vivo studies underscored the importance of preventing early inflammation and cell-recruitment to avoid islet graft loss in islet transplantation. Long-term survival of porcine islets in NHPs was shown to be feasible using T-cell-directed immunosuppression, including anti-CD154 mAb.

The need for an unlimited source of islets and a safer method of immunosuppression has limited the widespread application of islet transplantation. To remedy the shortage of donor tissue, xenotransplantation of neonatal porcine islets (NPI) has been proposed. In this study we sought to determine if combining co-transplantation of NPI with Sertoli cells (SC) with a short-term monoclonal antibody (mAb) therapy would prevent NPI xenograft rejection. We hypothesize that this combination of treatments will lead to long-term NPI xenograft survival. Our result show a significant increase in the proportion of mice achieving long-term graft survival compared to untreated mice transplanted with NPI alone, as 7/7 mice treated with anti-LFA-1 mAb (p=0.001), 7/8 mice treated with anti-CD154 mAb (p=0.003), and 4/9 mice treated with anti-CD45RB mAb (p=0.020) achieved and maintained normoglycemia long-term. Therefore, we conclude that the combination of mAb therapy with SC is highly efficacious in preventing NPI xenograft rejection. / Experimental Surgery

Amyloid is defined as extracellular protein aggregates with a characteristic fibrillar ultra-structure, Congo red affinity and a unique x-ray diffraction pattern. At present, 25 different human amyloid fibril proteins have been identified, and amyloid aggregation is associated with pathological manifestations such as Alzheimer’s disease, spongiform encephalopathy and type 2 diabetes. Amyloid aggregation triggers apoptosis by incorporation of early oligomers in cellular membranes, causing influx of ions. Amyloid is the only visible pathological islet alteration in subjects with type 2 diabetes, and islet amyloid polypeptide (IAPP) is the major islet amyloid fibril component. IAPP is produced by beta-cells and co-localized with insulin in the secretory granules. Both peptides are synthesised as pro-molecules and undergo proteolytic cleavage by the prohormone convertase 1/3 and 2. Although IAPP is the main amyloid constituent, both proIAPP and proIAPP processing intermediates have been identified in islet amyloid. The aim of this thesis was to study the role of impaired processing of human proIAPP in early islet amyloidogenesis. Five cell lines with individual processing properties were transfected with human proIAPP and expression, aggregation and viability were studied. Cells unable to process proIAPP into IAPP or to process proIAPP at the N-terminal processing site accumulated intracellular amyloid-like aggregates and underwent apoptosis. Further, proIAPP immunoreactivity was detected in intracellular amyloid-like aggregates in betacells from transgenic mice expressing human IAPP and in transplanted human beta-cells. ProIAPP was hypothesized to act as a nidus for further islet amyloid deposition, and to investigate this theory, amyloid-like fibrils produced from recombinant IAPP, proIAPP and insulin C-peptide/A-chain were injected in the tail vein of transgenic mice expressing the gene for human IAPP. Pancreata were recovered after 10 months and analysed for the presence of amyloid. Both IAPP and proIAPP fibrils but not des-31,32 proinsulin fibrils, caused an increase in affected islets and also an increase of the amyloid amount. This finding demonstrates a seeding capacity of proIAPP on IAPP fibrillogenesis. IAPP has been known for some time to trigger apoptosis in cultured cells, and a novel method for real time detection of apoptosis in beta-cells was developed. Aggregation of recombinant proIAPP and proIAPP processing intermediates were concluded to be inducers of apoptosis as potent as IAPP fibril formation. From the results of this study, a scenario for initial islet amyloidogenesis is proposed. Initial amyloid formation occurs intracellularly as a result of alterations in beta-cell processing capacity. When the host cell undergoes apoptosis intracellular proIAPP amyloid becomes extracellular and can act as seed for further islet amyloid deposition.

Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 151-168). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 196-214.

Ce travail explore l’impact des microparticules (MPs) sur la réponse vasculaire dans les conditions d’ischémie reperfusion (IR) associées à la dysfonction du greffon. Il décrit les effets pro-sénescents paracrines de MPs d’origine endothéliales (EMPs) et leucocytaires (LMPs) dans un modèle de communication intercellulaire et sur l’îlot pancréatique de rat, qui constitue une entité fonctionnelle multicellulaire. Nos résultats indiquent que les LMPs induisent une sénescence endothéliale prématurée associée à une dysfonction endothéliale, identifiée dans les cellules et les anneaux d’artères coronaires. De plus, cette dysfonction est caractérisée par une signalisation redox sensible pro-inflammatoire et un phénotype thrombogénique avec la libération secondaire d’EMPs pro-coagulantes. La deuxième étude indique que les EMPs pro-sénescentes provoquent une sénescence prématurée des îlots pancréatiques et altèrent leur fonctionnalité sans affecter leur viabilité. De ce fait, les MPs sont des cibles thérapeutiques intéressantes pour limiter l’IR et la dysfonction du greffon, notamment en greffe d’îlots pancréatiques. / This work explores the impact of microparticles (MPs) on vascular response under conditions of ischemia reperfusion (IR) associated with graft dysfunction. It describes the pro-senescent paracrine effects of endothelial- (EMPs) and leukocyte-derived MPs (LMPs) in a cellular cross-talk model and in rat pancreatic islets, which are multicellular functional units. Our results indicate that LMPs induce premature endothelial senescence associated with endothelial dysfunction, identified in coronary artery rings and endothelial cells. In addition, such endothelial dysfunction is characterized by a pro-inflammatory redox-sensitive signaling and a thrombogenic phenotype typified by a secondary release of pro-coagulant EMPs. The second study indicates that pro-senescent EMPs cause the premature senescence of islets and impair their functionality without affecting their viability. As a result, MPs are promising therapeutic targets for limiting IR and graft dysfunction, particularly in pancreatic islets transplantation.

Made available in DSpace on 2015-07-22T20:50:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-25 / O envelhecimento está associado à alteração da sensibilidade da célula β pancreática aos nutrientes resultando em intolerância à glicose e desenvolvimento do Diabetes mellitus (DM) do tipo 2 (DM 2). Neste estudo verificamos a função das células β no envelhecimento por meio da caracterização da sinalização do Ca2+, morfofisiologia mitocondrial e do estado redox de ilhotas pancreáticas de ratas senescentes com 24-25 meses de idade. Ilhotas isoladas de ratas senescentes secretaram menos insulina na presença de glicose. O influxo de Ca2+ em resposta à despolarização foi menor em ilhotas de ratos senescente em relação à ilhotas de ratas adultas. Ainda, a secreção de insulina potencializada pelo agente colinérgico carbacol (Cch) foi menor no grupo senescente. Ilhotas senescentes apresentaram menor produção de NAD(P)H em resposta à glicose, bem como menor potencial elétrico mitocondrial (ΔΨm) e maior acúmulo de espécies reativas de oxigênio (EROs) quando comparado ao grupo adulto. A análise das células β que compõem a ilhota pancreática, por meio de microscopia eletrônica, demonstrou que o grupo senescente apresenta grande quantidade de lisossomos e corpos residuais, e, alterações morfológicas nas mitocôndrias com cristas mitocondriais modificadas quando comparadas ao grupo adulto. Além disso, foi evidenciada a presença de invaginações nos envoltórios nucleares, que são indicativos de apoptose, nas células do grupo senescente. Contudo, a análise da taxa de apoptose por TUNEL não demonstrou alterações entre o grupo senescente e adulto. Portanto, o envelhecimento reduz a capacidade secretória da célula  pancreática frente à glicose e ao estímulo colinérgico. Estes efeitos estão relacionados a alterações na morfofisiologia mitocondrial das células que compõem à ilhota pancreática resultando em déficit metabólico e também maior geração de EROs. Estes efeitos juntamente com a redução da entrada de Ca2+ quando há despolarização e menor potencialização da secreção frente ao estímulo colinérgico, resultaram em prejuízos no adequado acoplamento estímulo/secreção das células . / Aging is associated with changes in pancreatic β cells sensitivity to nutrients resulting in glucose intolerance and diabetes mellitus (DM) type 2 (DM 2) development. Here the study cells function Ca2+ signaling, mitochondrial morphophysiology and redox state of isolated islets from senescent rats with 24-25 months old. Adult rats wister 4-5 months old were used as controls. Islets from senescent rats secreted less insulin in the presence of glucose. The influx of Ca2+ in response to depolarization is smaller in senescent islets when compared to adult islets. Also, the increase in insulin secretion induced by the cholinergic agent carbachol (Cch) was lower in the senescent islets. In addition, from senescent rats showed lower NAD (P) H production in response to glucose, decreased mitochondrial electric potential (ΔΨm) and a higher accumulation of the reactive oxygen species (ROS) when compared to islets from adult rats. Electron microscopy showed that the β cells from senescent islets presented higher lysosomes and residual bodies density, and changes in the morphology of the mitochondria, that showed alterations in the mitochondrial cristae when compared to adult group. Furthermore, nuclear invaginations were observed in cells of senescent group indicate apoptosis in this group. However, the rate of apoptosis analysed by TUNEL methodology showed no changes between the groups. In conclusions, the secretory capacity of the pancreatic β cells in the presence of glucose and the cholinergic stimulation were decreased in aging, these effects were associated with related mitochondrial morphophysiology dysfunction that result in decreased metabolism and increased ROS production. These effects together with the lower Ca2+ influx in response to depolarization and lower potentiation of the secretion in repose to the colinergic stimulus, results a diruption on the β cells stimulus/secretion coupling. / TEDE / BV UNIFESP: Teses e dissertações