<p>Clinical islet transplantation is an emerging procedure to cure type 1 diabetes. The graft is implanted by infusion into the liver through the portal vein. A major obstacle that still needs to be overcome is the requirement for islets from multiple donors to achieve insulin independence. </p><p>An innate inflammatory reaction, the IBMIR, is elicited when islets are exposed to blood. The IBMIR has been described as a clotting reaction culminating in disruption of islet morphology and is a plausible cause for loss of tissue during the early post-transplant period. </p><p>In this thesis, the underlying mechanisms of the IBMIR were characterized. The IBMIR was for the first time demonstrated in patients undergoing an islet transplant, and a number of clinically applicable strategies to limit this reaction were identified.</p><p>The thrombin inhibitor melagatran completely blocked the IBMIR in an <i>in vitro</i> tubing blood loop system, indicating that thrombin is the driving force in the reaction. Interestingly, islets were shown to produce and secrete tissue factor (TF), the physiological trigger of coagulation. Inactivated FVIIa, a specific inhibitor of TF, successfully blocked initiation of the IBMIR. An alternative approach to limit the IBMIR was to pre-treat islets in culture prior to transplantation. Nicotinamide added to the culture medium effectively decreased the level of TF in human islets. Infiltration of immune cells, also a part of the IBMIR, was characterized in detail. The predominant cell types infiltrating the islets were neutrophilic granulocytes and, to a lesser degree, monocytes. Both cell types may exert direct cytotoxic effects, and the antigen-presenting monocytes may also be important for directing the specific immune system to the site of inflammation. </p><p>These findings have provided new insight into the nature of the IBMIR and offer several new strategies to improve the outcome of clinical islet transplantation.</p>

Clinical islet transplantation is an emerging procedure to cure type 1 diabetes. The graft is implanted by infusion into the liver through the portal vein. A major obstacle that still needs to be overcome is the requirement for islets from multiple donors to achieve insulin independence. An innate inflammatory reaction, the IBMIR, is elicited when islets are exposed to blood. The IBMIR has been described as a clotting reaction culminating in disruption of islet morphology and is a plausible cause for loss of tissue during the early post-transplant period. In this thesis, the underlying mechanisms of the IBMIR were characterized. The IBMIR was for the first time demonstrated in patients undergoing an islet transplant, and a number of clinically applicable strategies to limit this reaction were identified. The thrombin inhibitor melagatran completely blocked the IBMIR in an in vitro tubing blood loop system, indicating that thrombin is the driving force in the reaction. Interestingly, islets were shown to produce and secrete tissue factor (TF), the physiological trigger of coagulation. Inactivated FVIIa, a specific inhibitor of TF, successfully blocked initiation of the IBMIR. An alternative approach to limit the IBMIR was to pre-treat islets in culture prior to transplantation. Nicotinamide added to the culture medium effectively decreased the level of TF in human islets. Infiltration of immune cells, also a part of the IBMIR, was characterized in detail. The predominant cell types infiltrating the islets were neutrophilic granulocytes and, to a lesser degree, monocytes. Both cell types may exert direct cytotoxic effects, and the antigen-presenting monocytes may also be important for directing the specific immune system to the site of inflammation. These findings have provided new insight into the nature of the IBMIR and offer several new strategies to improve the outcome of clinical islet transplantation.

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.

Background: Glucose homeostasis is tightly regulated by hormones secreted within the pancreatic islets of Langerhans. The most important are insulin and glucagon produced by beta and alpha cells respectively. Changes in beta cell mass and/or their functional deficit can lead to hyperglycemia, a major hallmark of both type 1 (T1D) and type 2 (T2D). Moreover, a dysregulation in glucagon secretion is thought to also play a major role in patients with diabetes, suggesting a failure in the counterregulatory mechanisms of glucose homeostasis in disease pathogenesis. Dysfunction at the alpha cell level in T1D manifests are blunt glucagon response to low glucose levels, which can cause severe hypoglycemic events in patients with T1D. Furthermore, exaggerated glucagon responses to glucose or amino acid intake significantly contributes to dysglycemia in both T1D and T2D patients. Most of our knowledge about glucagon and alpha cell physiology in the human setting was generated using in vivo systemic assessments or in vitro investigations of isolated human islets or dispersed single cells. Despite the increasing knowledge regarding alpha cells and glucagon biology, the underlying mechanisms of alpha cell dysfunction are still uncertain. Studies on alpha cell physiology were hindered by limited human tissue accessibility, technical methodologies and translational value of findings from rodents to humans. To fill the gap between the currently available in vivo and in vitro approaches and a more precise understanding of mechanisms of diabetes pathogenesis detailed investigation of islet cells within their native environment is needed. Aim The overall objective of this thesis was characterize alpha cell function in diabetes pathogenesis. To this end, the human pancreas slice preparation would to be adapted and advanced for the study of alpha cell physiology. These adjustments would be then used to investigate changes in alpha cell mass and function in T1D and T2D. Methods: Pancreas tissue slices were prepared from donor organs with and without T1D and from tissue donors after pancreatectomy at different stages of T2D. Immunofluorescent staining with subsequent 3D morphometry was used to quantify alpha cell volumes from 120μm thick tissue slices. Furthermore, human tissue slices were subjected to dynamic slice perifusion for the assessment of glucagon and insulin secretion kinetics in response to specific stimuli. Finally, functional and morphometrical analysis was performed on the same tissue slices to enable direct correlation of glucagon secretion and alpha cell volume in a subset of cases in the context of T1D. Results: Here we developed a semi-automatic 3D approach to quantify total endocrine cell volumes within a given volume of pancreas tissue. In addition, we established an in situ method for dynamic insulin release measurements from islets preserved in their native environment. We successfully modified this protocol to allow the measurement of glucagon release in slices from organ donors. After further optimizations, we were additionally able to also measure alpha cell function from surgical specimens after pancreatectomy. To gain insight into alpha cell pathophysiology in T1D we investigated alpha cell volume in donor organs with different disease duration and age at onset. Alpha cell volumes in slices of individuals with T1D did not show a dramatic change (neither increase nor decrease) in comparison to slices generated from non-diabetic (ND) pancreata. Furthermore, functional assessment of glucagon release using a specific stimulation protocol for alpha cells suggests preserved stimulatory capacity of these cells in slices from autoantibody positive donors. Interestingly, this is also the case in the so far studied slices from donors with different durations of diabetes. Nevertheless, normalization of secreted glucagon to the total alpha cell mass within the slice indicated reduced glucagon release in the here investigated two cases of T1D. In the context of T2D, 3D morphometrical analysis revealed that overall endocrine cell volume, including alpha cell volume, is maintained in our cohort of IGT and T2D individuals. Glucagon release can also be measured in tissue procured from patients undergoing pancreatectomy, given the presence of amino acids in the perifusion media and increased trypsin inhibitors. While we provided proof of concept using tissue from ND individuals, we are confident that the approach will give valuable insight in different states of diabetes. Conclusion: These results demonstrate that human pancreas tissue slices represent a complementary platform to study alpha cell pathophysiology in both major types of diabetes. We provide evidence that this approach can be used to study alpha cell pathophysiology in T1D and T2D. Our preliminary data indicates no defect in the stimulatory capacity in slices from Aab+ and T1D donors, however more cases need to be investigated given the heterogeneous nature of the disease. We anticipate that the here proposed protocol for measurement of glucagon release from tissue slices will help us to gain insight in the role of alpha cells in diabetes pathophysiology. / Hintergrund: Die Aufrechterhaltung der Glukosehomöostase wird durch die Hormonsekretion der Langerhans’schen Inseln im Pankreas reguliert. Die wichtigste Rolle hierbei spielen die Insulin-produzierenden Betazellen und die Glukagon-produzierenden Alphazellen. Der Verlust der Betazellmasse und/oder der Funktion kann zur Entwicklung einer Hyperglykämie führen, die ein Hauptmerkmal des Typ 1 (T1D) und Typ 2 (T2D) Diabetes ist. Darüber hinaus wird vermutet, dass auch der Mechanismus der Gegenregulierung durch die Sekretion von Glukagon eine wichtige Rolle in der Pathogenese des Diabetes spielt. Während eine fehlende Glukagonsekretion zu schweren hypoglykämischen Phasen bei Typ 1 Diabetikern führen kann, geht man zusätzlich davon aus, dass eine erhöhte Reaktivität von Alphazellen sowohl auf Glukose als auch Aminosäuren ebenfalls zum Verlust der Glukosehomöostase im T1D und T2D beitragen kann. Trotz der stetig wachsenden Erkenntnisse über die Physiologie der Alphazellen, die vor allem durch systemische Untersuchungen in vivo oder an isolierten Langerhans’schen Inseln und Einzelzellen in vitro durchgeführt wurden, sind die zugrundeliegenden Mechanismen für deren Fehlfunktion beim Menschen noch nicht eindeutig aufgeklärt. Dies beruht hauptsächlich auf nur bedingt vorhandenem humanem Gewebe, technischen Schwierigkeiten bei der Isolation der Zellen, sowie der nur bedingten Vergleichbarkeit zu Studien in Nagern. Um diese Wissenslücken zwischen in vivo und in vitro Studien schließen zu können, ist es notwendig detaillierte Untersuchungen der Zellen unter nahezu physiologischen Bedingungen und in der nativen Umgebung der Pankreas in situ durchzuführen, um Alphazell-spezifische Mechanismen in der Diabetespathogenese genauer beleuchten zu können. Ziele: Ziel dieser Dissertation war es, durch Anpassung und Weiterentwicklung der Technik zur Gewinnung von Gewebeschnitten des humanen Pankreas, die Funktion der Alphazellen sowohl unter physiologischen Bedingungen als auch in der Entwicklung des T1D und T2D genauer zu charakterisieren. Methoden: Zur Untersuchungen von Alphazellen in Gewebeschnitten in situ wurden Gewebestücke sowohl von Organspendern mit und ohne T1D, sowie von metabolisch charakterisierter Patienten in verschiedenen Stadien der T2D Pathogenese nach einer Pankreatektomie verwendet. Die aus dem Gewebe gewonnenen 120 μm dicken Schnitte wurden zum einen für immunhistochemischer Färbungen verwendet, die eine 3-dimensionale morphometrische Analyse der Alphazellmasse ermöglichen. Ferner wurden Schnitte zur Ermittlung der Kinetik von Glukagon- und Insulinsekretion nach Stimulation mittels Perifusion benutzt. Schließlich wurden sowohl die morphologischen als auch funktionellen Analysen auf denselben Gewebeschnitten durchgeführt, um die Funktion der Alphazellen mit deren Masse besser korrelieren zu können. Ergebnisse: Zusätzlich zur Mitentwicklung eines halbautomatisierten Verfahrens zur 3D Analyse von endokrinen Zellvolumina in Gewebeschnitten des Pankreas wurde die bereits vorhandene Methode zur Messung der Insulinsekretionskinetik weiterentwickelt. Außerdem erfolgte die Etablierung adäquater Protokolle zur Messung der Glukagonsekretion in humanen Gewebeschnitten, die im Kontext beider Diabetestypen verwendet wurden. Um einen besseren Einblick in die T1D Pathogenese zu erhalten, wurde das Alphazellvolumen- und die Funktion in Gewebeschnitten von Organspendern mit unterschiedlichem Diabetes Verlauf (Alter bei Diagnose, Dauer seit Diagnose) mit nicht-diabetischen, aber Autoantikörper-positiven Spendern und nicht-diabetische Kontrollen verglichen. In Bezug auf das Alphazellvolumen waren zwischen den einzelnen Gruppen keine Unterschiede zu erkennen, die auf Veränderungen in der Entwicklung und Manifestation des T1D hinweisen. Darüber hinaus ergab die funktionelle Analyse, dass die Glukagonsekretion in nicht-diabetischen, Autoantikörper-positiven Spendern erhalten bleibt. Dies konnte zusätzlich auch in den bisher untersuchten Geweben von Typ 1 Diabetikern nachgewiesen werden, obwohl die Volumen-normalisierte Sekretion auf eine geringere Glukagonausschüttung hindeutet. Im Hinblick auf die T2D Pathogenese konnte bei der 3D Morphometrie von Nichtdiabetikern, Patienten mit beeinträchtigter Glukosetoleranz und Typ 2 Diabetikern keinerlei Unterschiede in den endokrinen Zellvolumina festgestellt werden. Durch die Anpassung der Konditionen für die Perifusion von reseziertem Gewebe konnte bei Nichtdiabetikern die erfolgreiche Messung der Glukagonsekretion gezeigt werden und ermöglicht zukünftig auch die Untersuchung einer möglichen Alphazelldysfunktion in der Entwicklung eines T2D. Schlussfolgerung: Die Ergebnisse dieser Arbeit zeigen, dass die etablierte Plattform zur morphologischen und funktionellen Analyse humaner Pankreasgewebeschnitte in situ eine wichtige Rolle in der Untersuchung der Alphazellen in der Diabetes-Pathogenese spielt. Die bisher erhobenen Daten zur Untersuchung des T1D haben gezeigt, dass die Kapazität der Glukagonsekretion nicht signifikant verändert ist. Aufgrund des heterogenen Krankheitsverlaufs beider Diabetesformen ist es jedoch notwendig Gewebe von einer größeren Anzahl an Spendern/Patienten zu untersuchen, um einen besseren Einblick in die Rolle der Alphazellen in der Entstehung des Diabetes zu erhalten.

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(English) Perfluorocarbons (PFC) are hydrocarbons in which some or all of the hydrogen atoms are replaced with fluorine. PFC have a very high capacity for dissolving oxygen. They are chemically and biologically inert. The most successful clinical application of PFC is the "two-layer method" for pancreas preservation before islet isolation. The two-layer organ preservation method (TLM) is based on oxygenated perfluorocarbon overlaid with University of Wisconsin (UW) solution. In experiment it has been successfully used for heart and intestine transplantation. We tested whether this technique would prevent tissue damage and improve results of kidney, pancreas and islets of Langerhans transplantation with prolonged ischemia time in an experimental model of syngenic rats. In kidney and islets of Langerhanse transplantation model we used TLM preservation method. In pancreas transplantation model we used perfluorohexyloctane (PFH) as a new generation of less lipophilic PFC. 1. Kidneys were stored for 24 hours either in UW solution (n = 16), with TLM (n = 16) or transplanted immediately (control group, n = 12). In half of the animals, survival was observed and in the other animals grafts were procured for semiquantitative histological scoring and TUNEL apoptosis assessment 24 h after transplantation....