Diabetes is a debilitating disease affecting millions of people worldwide. The transplantation of insulin-producing, pancreatic islet cells has been an extensively explored approach for the treatment of Type 1 Diabetes. However, the need for a multi-donor source, the strong host immune responses, and a life-long immunosuppressive therapy regimen limits the widespread applicability of islet transplantation. Encapsulation of islet cells within a semi-permeable biomaterial as a means to mask transplanted cells from the host has been shown to be a viable option for the protection of islets upon transplantation. Recent advancements, incorporating additional knowledge of biomaterials, have revitalized the field of islet encapsulation. This thesis work focused on both micro- and nano-scale encapsulation techniques. Initially, a novel, covalently linked alginate-poly(ethylene glycol) (PEG), termed XAlginate-PEG, microcapsule was evaluated, and was shown to exhibit superior stability over traditional ionically bound alginate microcapsules. The XAlginate-PEG capsules exhibited a 5-fold decrease in osmotic swelling than traditional alginate microcapsules, and remained completely intact upon chelation of ionic interactions. In addition, in vitro study of the novel polymer matrix showed high compatibility with mouse insulinoma cell lines, rat and human islets. Furthermore, no disruption in islet function was observed upon encapsulation. The second study of this thesis work focused on the nano-scale encapsulation of islets with a single layer PEG coating. A PEG polymer was grafted directly on the collagen matrix of the islet capsule to form a stable amide bond. PEGylation of the islet cells was shown to camouflage inflammatory agents, such as tissue factor (TF), present on the surface of the islet, while maintaining islet morphology and function. In summary, PEG dampened coagulation cascade activation, and concealed activated factor X (afX) generation under pro-inflammatory culture conditions. The present findings contribute to the field of cellular encapsulation, both in the fabrication of novel encapsulation techniques and the evaluation of nano-scale coatings. The future potential of this research includes the attenuation of immune responses to transplanted cells, elimination of continuous immunosuppression, and provide flexibility in cell source. Furthermore, the platforms evaluated in this thesis are generalized for all cell types, thereby permitting translation of techniques to alternative cellular therapies.

CD4+CD25+FoxP3+ regulatory T cells (Treg) possess the capacity to modulate both adaptive and innate immunity. Due to their suppressive nature, Treg cells have been studied and tested in a variety of scenarios in an attempt to ameliorate undesired immune responses. While graft versus host disease (GVHD) has in fact emerged as the first clinical application for human Treg cells (Riley et al. 2009), equally important are issues concerning hematopoietic engraftment and immune reconstitution. Currently, little is known about the effect(s) that regulatory T cells may exert outside the immune system in this context. Based on cytokine effector molecules they can produce we hypothesized that Treg cells could regulate hematopoietic phenomena. The studies portrayed in this dissertation demonstrate that Treg cells can differentially affect the colony forming activity of myeloid and erythroid progenitor cells. In-vitro as well as in-vivo findings demonstrate the ability of Tregs to inhibit and augment the differentiation of primitive and intermediate myeloid (interleukin (IL)-3 driven) and late erythroid (erythropoietin driven) hematopoietic progenitor cells, respectively. The inhibitory and enhancing affects appeared to be mediated by independent pathways, the former requiring cell-cell contact, major histocompatibility complex (MHC) class II expression on marrow cells and involving transforming growth factor beta (TGF-beta), whereas the latter required interleukin (IL)-9 and was not contact dependent. Strikingly, we observed that in addition to regulating hematopoietic activity in normal primary BM cells, Tregs were also capable of suppressing colony forming activity by the myelogenous leukemia cell line NFS-60. Furthermore, studies involving endogenous Treg manipulations in-situ (i.e. depletion of these cells) resulted in elevated overall myeloid colony activity (CFU-IL3) and diminished colony numbers of erythroid precursors (CFU-E) in recipients following BMT. Consistent with these results, it was found that upon co-transplant with limiting numbers of bone marrow cells, exogenously added Treg cells exert in-vivo regulation of myeloid and erythroid CFU activity during the initial weeks post-transplantation. This regulation of hematopoietic activity by freshly generated Tregs upon transplantation led to the elaboration of a second hypothesis; following lethal total body irradiation (TBI) the host microenvironment facilitates regulatory T cell activation/effector function. Substantial evidence has accumulated in support of this hypothesis, for example we demonstrate up-regulation of surface molecules such as GARP and CD150/SLAM, which have been previously reported as indicators of Treg activation following TCR signaling and co-stimulation, occurs in donor (reporter) Treg populations. Acquisition of an activated phenotype and hence of effector/modulatory function is consistent with the previous in-vivo observations, indicating that both recipient and donor Treg cells can influence hematopoietic progenitor cell activity post-transplant. Finally, the present studies may be of great relevance in the emerging field of Treg cell based immunotherapy for prevention and/or treatment of HSCT complications.

A. INTRODUCTION A.1. The sympathetic nervous system. A.1.1. General considerations and historical perspective. A.1.1.1. Historical perspective A.1.1.2. Reflex regulation of the autonomic nervous system A.1.1.3. Central control of the autonomic nervous system A.1.1.4. Sympathetic and parasympathetic components of the autonomic nervous system A.1.1.5. Organisation of the sympathetic nervous system A.1.1.6. Functions of the sympathetic nervous system A.1.1.7. Neurotransmitters of the sympathetic nervous system A.1.1.8. Neurotransmitter secretion at effectors organ synapse A.1.1.9. Adrenoreceptors A.1.2. Control mechanisms A.1.2.1. Aortic arch and carotid baroreceptors A.1.2.2. Low pressure baroreceptors A.1.2.3. Chemoreceptors A.1.2.4. Effects of exercise on sympathetic nervous system activation A.1.2.5. Effects of left ventricular dysfunction on sympathetic nervous system activation A.1.2.6. Effects of right ventricular dysfunction and heart transplantation on sympathetic nervous system activity A.2. Methodological considerations. A.2.1. Assessment of sympathetic activity in humans A.2.2. Circulating catecholamines A.2.3. Microneurography A.3. Ergospirometry A.3.1. Several aspects of physiology of exercise A.3.2. Principles of exercise testing A.3.3. Exercise ventilation A.4. Assessment of chemoreceptor regulation in humans A.4.1. Peripheral chemoreceptor inhibition A.4.2. Peripheral and central chemoreceptor activation A.5. Brief summary of still unresolved questions A.5.1. Pulmonary arterial hypertension A.5.2. Heart transplantation B. SYMPATHETIC CONTROL IN PULMONARY ARTERIAL HYPERTENSION B.1. Hypothesis tested B.2. Study populations B.2.1. Study investigating sympathetic activity in PAH patients B.2.2. Study investigating the effects of atrial septostomy on MSNA in PAH patients B.3. Material, methods and study protocols B.3.1. Particular measurements in the study investigating sympathetic activity in PAH patients B.3.2. Particular measurements in the study investigating effects of atrial septostomy on MSNA in PAH patients B.4. Sympathetic nervous activity in PAH and effects of disease severity B.5. Effects of chemoreflex activation B.6. Effects of atrial septostomy C. SYMPATHETIC CONTROL AFTER HEART TRANSPLANTATION C.1. Hypothesis tested C.2. Patient population C.3. Material and methods C.4. Effects of chemoreflex activation on sympathetic activity and blood pressure C.5. Effects of chemoreflex activation on exercise intolerance D. DISCUSSION D.1. Sympathetic nervous system activation in patients with pulmonary arterial hypertension D.2. Effects of atrial septostomy on sympathetic nervous system activation D.3. Chemoreceptors in heart transplant recipients D.3.1. Peripheral chemoreceptors deactivation D.3.2. Peripheral and central chemoreceptors sensitivity E. CONCLUSIONS F. REFERENCE LIST G. ANNEXES G.1. Publications G.1.1. Velez-Roa and Ciarka et al, Increased sympathetic nerve activity in pulmonary artery hypertension, Circulation. 2004 Sep 7;110(10):1308- 12. G.1.2. Ciarka et al, Atrial septostomy decreases sympathetic overactivity in pulmonary arterial hypertension, Chest. 2007 Jun;131(6):1831-7. G.1.3. Ciarka et al, Effects of peripheral chemoreceptors deactivation on sympathetic activity in heart transplant recipients. Hypertension. 2005 May;45(5):894-900. G.1.4. Ciarka et al, Increased peripheral chemoreceptors sensitivity and exercise ventilation in heart transplant recipients. Circulation. 2006 Jan 17;113(2):252-7. G.2. Annexe thesis title. G.3. Brief summary in French of described research

Transplantation of insulin producing cells is currently the only cure for type 1 diabetes. However, even though the Edmonton protocol markedly increased the success rate of pancreatic islet transplantation, the long term insulin independence is still very poor. An adequate engraftment is critical for islet graft survival and function. In the present thesis, isolated islet endothelial cells were found to have a low proliferatory and migratory capacity towards vascular endothelial growth factor (VEGF), but this could be reversed by using neutralizing antibodies to the angiostatic factors thrombospondin-1, endostatin or alpha1-antitrypsin. In the adult islet endothelial cell, VEGF may act as a permeability inducer more than an inducer of angiogenesis. p38 MAP kinase activity has been shown to serve as a switch between these properties of VEGF. Inhibition of p38 MAP kinase by daily injections of SB203580 in the early posttransplantation phase lead to a redistribution of the islet graft blood vessels from the stroma into the endocrine tissue and this was accompanied by a higher oxygen tension. Besides transports of oxygen and nutrients, beta-cells may require signals from the endothelial cells for their growth and differentiation. It was demonstrated that islet endothelial cells secrete factors, including laminin, that have positive effects on beta-cell insulin release and insulin content. Our results suggest that improved revascularization of transplanted islets may be achieved by either inhibition of angiostatic factors, or by blocking p38 MAPkinase activity, in the implanted tissue. Islet endothelial cells have a supportive paracrine role for beta-cells that might be hampered by the normally poor revascularization.

BACKGROUND: Organ transplantation is one of the best modalities for treating fatal organ failure. Despite the success of this procedure, an increasing incidence of cancer in this population has drawn the attention of public health officials in recent years. OBJECTIVES: The overall objective of this study is to conduct a detailed examination of adverse health outcomes among Canadian organ transplant recipients, with an emphasis on cancer incidence and mortality. METHODS: This project employed a retrospective cohort follow-up study design, whereby Canadian Organ Replacement Registry records were linked to the Canadian Mortality Database and the Canadian Cancer Registry Database. The study population consisted of more than 16,000 solid organ transplant recipients registered between January 1, 1981 and December 31, 1998. This study was designed to assess the risks of developing cancer, overall and site-specific, in transplant recipients in comparison to the general Canadian population using Standardized Incidence Ratios (SIR), Standardized Mortality Ratios (SMR), and Proportionate Mortality Ratios (PMR). In addition, Cox and logistic models were used to assess the effects of various risk factors on cancer incidence and mortality in transplant sub-populations, while cumulative incidence was used to study the patient survival pattern. Lastly, Population Attributable Risk (PAR) was used to quantify the impact of organ transplantation on cancer incidence and mortality. RESULTS: Among major causes of death, the highest PMRs are due to genitourinary diseases, followed by endocrine, nutritional and metabolic diseases, and infectious diseases. SIRs indicate that cancer incidence and mortality were relatively lower than that observed for other major causes of death, and slightly higher than that observed in the general Canadian population. Lastly, logistic regression results indicate that age, year of surgery, and smoking status were significant risk factors in mortality due to all causes, while the Cox regression model shows that age, sex and year of surgery were significant risk factors for cancer incidence. Overall, the PAR in this cohort was very minimal, indicating that the risk in mortality and cancer incidence due to organ transplantation is negligible. CONCLUSION: Life threatening diseases such as those of the genitourinary system, as well as endocrine, nutritional and metabolic diseases and infectious diseases are leading causes of death. Future research should be directed at ways of reducing incidence and subsequent mortality due to these causes.

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.

No description available.

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.

BACKGROUND: Organ transplantation is one of the best modalities for treating fatal organ failure. Despite the success of this procedure, an increasing incidence of cancer in this population has drawn the attention of public health officials in recent years. OBJECTIVES: The overall objective of this study is to conduct a detailed examination of adverse health outcomes among Canadian organ transplant recipients, with an emphasis on cancer incidence and mortality. METHODS: This project employed a retrospective cohort follow-up study design, whereby Canadian Organ Replacement Registry records were linked to the Canadian Mortality Database and the Canadian Cancer Registry Database. The study population consisted of more than 16,000 solid organ transplant recipients registered between January 1, 1981 and December 31, 1998. This study was designed to assess the risks of developing cancer, overall and site-specific, in transplant recipients in comparison to the general Canadian population using Standardized Incidence Ratios (SIR), Standardized Mortality Ratios (SMR), and Proportionate Mortality Ratios (PMR). In addition, Cox and logistic models were used to assess the effects of various risk factors on cancer incidence and mortality in transplant sub-populations, while cumulative incidence was used to study the patient survival pattern. Lastly, Population Attributable Risk (PAR) was used to quantify the impact of organ transplantation on cancer incidence and mortality. RESULTS: Among major causes of death, the highest PMRs are due to genitourinary diseases, followed by endocrine, nutritional and metabolic diseases, and infectious diseases. SIRs indicate that cancer incidence and mortality were relatively lower than that observed for other major causes of death, and slightly higher than that observed in the general Canadian population. Lastly, logistic regression results indicate that age, year of surgery, and smoking status were significant risk factors in mortality due to all causes, while the Cox regression model shows that age, sex and year of surgery were significant risk factors for cancer incidence. Overall, the PAR in this cohort was very minimal, indicating that the risk in mortality and cancer incidence due to organ transplantation is negligible. CONCLUSION: Life threatening diseases such as those of the genitourinary system, as well as endocrine, nutritional and metabolic diseases and infectious diseases are leading causes of death. Future research should be directed at ways of reducing incidence and subsequent mortality due to these causes.

Die orthotope Lebertransplantation (OLT) ist ein etabliertes Standardtherapieverfahren von Endzuständen verschiedenster Lebererkrankungen. Bei Patienten mit Lebererkrankungen kommt es zu einer deutlichen Reduktion der hepatischen Metabolisierung und Elimination von verschiedensten Medikamenten. Fentanyl ist das Opioid der Wahl im Rahmen der Anästhesie bei Patienten mit Lebererkrankungen. Die Pharmakokinetik von Fentanyl ist besonders in der anhepatischen Phase durch einen Ausfall der hepatischen Elimination gekennzeichnet, sodass es zu hohen Plasmakonzentrationen von Fentanyl kommen kann. Besonders bei der Fentanylinfusion bis zur Reperfusion kann dies zu einer verzögerten Extubation führen, welche im Rahmen des Fast-Track-Verfahrens vermieden werden sollte. Hauptanliegen unserer Studie ist der Vergleich zweier Infusionsregimes für das Opioid Fentanyl, nämlich der Beendigung der Fentanylapplikation mit Beginn der anhepatischen Phase (Studiengruppe) und zum Zeitpunkt der Reperfusion (Kontrollgruppe). Dazu wurden von 22 Patienten (Studiengruppe: n=10; Kontrollgruppe: n=12) intraoperativ zu neun definierten Messzeitpunkten (MZP) die Verläufe der arteriellen Fentanylplasmakonzentration, sowie von Parametern des Säure-Basen-Haushaltes und der hämodynamische Messwerte erfasst. Die Bestimmung der Fentanylkonzentration erfolgte mit Hilfe der Flüssigchromatographie mit Massenspektrometrie (SSQ 7000, Finnigan), der Säure-Basen-Haushalt mittels Blutgasanalyse (ABL 700, Radiometer Medical A/S, Kopenhagen). Hinsichtlich der Daten für MELD-Score, Diagnose und Gesamtoperationsdauer unterschieden sich die beiden Gruppen nicht signifikant voneinander. Im Verlauf der anhepatischen Phase lag die Fentanylkonzentration im Plasma in der Kontrollgruppe signifikant höher als in der Studiengruppe. Die gefundenen höheren Fentanylspiegel in der Kontrollgruppe sind Ausdruck einer nicht vorhandenen hepatischen Fentanylclearance in der anhepatischen Phase. Basierend auf unseren Daten wollen wir zu einer Überprüfung des Infusionskonzeptes von Fentanyl bei OLT anregen. Während einer OLT sollte im Hinblick auf das Fast-Track-Konzept über eine Beendigung der Fentanylzufuhr zum Beginn der anhepatischen Phase nachgedacht werden.