While cancer treatments have advanced for multiple cancers, pancreatic cancer remains a lethal cancer with few therapy options available. This is due to the inaccessibility of the tumor by surgical and thermal ablative means, high potential for chemoresistance and metastasis, and strongly immunosuppressive tumor microenvironment that makes new treatment measures ineffective in clinic. Irreversible electroporation (IRE) utilizes short, high voltage electrical pulses to form microlesions in cell membranes and induce cell death. While IRE has had significant impact in pancreatic cancer treatment in clinical trials, little is known on the biological and immunological effects of IRE on pancreatic cancer. By studying the effects of IRE on pancreatic tumor biology and the host immune system, I hypothesize I can identify potential combination therapy targets for IRE. I utilized in vitro, ex vivo, and in vivo animal models of both human and murine cancer to study the effects of IRE on pancreatic cancer progression and its potential for immunomodulation. My findings have shown that IRE can significantly delay cancer progression by inducing necroptosis in the tumor and altering the tumor microenvironment by increasing inflammatory signaling. IRE can also produce viable antigens for presentation to induce local and systemic immunosurveillance. However, these effects are limited by countering expression of programmed-cell death ligand 1 (PD-L1), a checkpoint protein that inhibits cytotoxic lymphocyte activity and allows the tumors to recur. The effects of IRE can therefore be expanded by multiple combination therapy approaches, such as chemotherapeutic application (potentially with nanoparticle packaging), PD-1/PD-L1 antibody immunotherapies, and small molecule inhibitors directed at tumor growth signaling that previously showed limited efficacy in clinic. / Doctor of Philosophy / While cancer treatments have advanced for multiple cancers, pancreatic cancer remains a lethal cancer with few therapy options available. This is due to limited surgical candidacy, resistance to chemotherapy, high potential for secondary tumor formation, and the cloaking of the tumor from the immune system that make new treatment measures ineffective in clinic. Irreversible electroporation (IRE) utilizes short, high voltage electrical pulses to form permanent pores in cell membranes and induce cell death. While IRE has had significant impact in pancreatic cancer treatment in clinical trials, little is known on how IRE affects pancreatic cancer biological or how it can alter the immune system. By studying the effects of IRE on pancreatic tumor biology and the host immune system, I hypothesize I can identify potential combination therapy targets for IRE. I utilized cell, tissue, and animal models of both human and mouse pancreatic cancer to study the effects of IRE on disease progression and its potential for inducing immune responses. My findings have shown that IRE can significantly delay cancer progression by inducing controlled inflammatory cell death in the tumor and altering the supportive cells populations in the tumor that allows for immune system recognition. IRE can also produce markers specific to the tumor for presentation to induce recognition of the primary tumor and secondary lesions in the body. However, these effects are limited by countering expression of programmed-cell death ligand 1 (PD-L1), a checkpoint protein that reduces immune cell activity and allows the tumors to recur. The effects of IRE can therefore be expanded by multiple combination therapy approaches, such as chemotherapeutic application (potentially with nanoparticle packaging), PD-1/PD-L1 antibody immunotherapies, and small molecule inhibitors directed at tumor growth signaling that previously showed limited efficacy in clinic.

La sténose aortique (SA) calcifiante est la troisième maladie cardiovasculaire en importance dans le monde. De surcroît, elle est au premier rang du palmarès des maladies valvulaires dans les pays industrialisés. Elle a longtemps été perçue comme étant une pathologie aux mécanismes dégénératifs passifs. Néanmoins, un nombre grandissant d'études ont démontré que cette maladie est active et régulée par diverses voies de signalisations. D'ailleurs, plusieurs facteurs de risques influencent le développement de la SA. Les mécanismes les plus importants, mais mal définis, qui sont impliqués dans la pathobiologie de la SA sont l'infiltration lipidique, l'inflammation, la fibrose et la calcification. Les liens qui unissent ces voies sont en investigations constantes. De meilleures connaissances vont permettre de découvrir une ou des avenues thérapeutiques contre le développement de la SA. D'une part, le système rénine-angiotensine (SRA) serait une cible thérapeutique possible contre le développement de la SA. Deux médicaments peuvent bloquer le SRA: les antagonistes des récepteurs de type 1 de l'angiotensine II (ARA) et les inhibiteurs de l'enzyme de conversion de l'angiotensine (IECA). Il est connu que l'activation du SRA est impliquée dans les processus d'inflammation, de fibrose et de remodelage des tissus de la valve aortique. D'autre part, la modulation de l'inflammation pourrait avoir des effets dans le développement de la SA. Cependant, son impact réel sur la progression active de la calcification dans le SA n'est pas encore établi. Dans un premier temps, nous avons démontré que la prise d'ARA et non d'IECA est reliée à une diminution du degré de remodelage, de fibrose, de certains marqueurs d'inflammation et de la progression de la SA. Dans un deuxième temps, nous avons démontré que l'inflammation est un régulateur actif de la SA. Elle est associée à des indices de sévérités hémodynamiques augmentés, à une haussée de la néovascularisation et à l'apparition de métaplasie osseuse. Pour terminer, il a été établi que la calcification est un élément crucial dans la développement de la SA. L'accumulation de dépôts calciques sur les feuillets de la valve aortique crée un obstacle au passage du sang. Parallèlement, dans l'os, le système ecto-purinergique est essentiel pour le maintient d'une balance efficace entre la formation et la résorption osseuse. Dans la SA, il n'a jamais été étudié en profondeur. Dans cette thèse, il a été montré que l'inhibition de l'ectonucléotide pyrophosphatase/phosphodiesterase 1 (ENPP1) favorise l'activation du récepteur à nucleotide, le P2Y2 ainsi que sa signalisation anti-apoptotique et anti-calcifiante. En somme, les travaux effectués pendant ce doctorat suggèrent que l'utilisation d'ARA et d'inhibiteurs des ectonucléotidases ENPP1 s'avèrent être des avenues thérapeutiques prometteuses dans le traitement de la SA.

La galectine-3 (Gal-3) appartient une famille de protéines liant les motifs p-galactosides grâce à un domaine conservé de reconnaissance des sucres. Cette lectine est impliquée à diverses étapes de l'immunité innée. Nous proposons que la Gal-3 démontre un potentiel pro-inflammatoire. L'injection de Gal-3 dans la poche d'air chez la souris a mené à un recrutement cellulaire important composé de neutrophiles, de macrophages et de quelques éosinophiles atteignant sont maximum à 6 h pour revenir à un niveau basai après une période de 24 h. Les cytokines sécrétées au niveau de la poche d'air ainsi que des fibroblastes incluaient le TNF-a, lTL-ip, 1TL-6, MIP-2, KC et MIP-la. La Gal-3 a aussi été en mesure d'activer la voie NF-kB in-vitro chez les fibroblastes. Cependant, la Gal-3 ne semble pas impliquée dans la phagocytose de pathogènes par les macrophages. En conclusion, la Gal-3 est pro-inflammatoire car elle active la voie NF-kB et cause le recrutement de neutrophiles sans leur être chimio-attractant

No description available.

Chow, Yin Sau Joyce. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 127-140). / Abstract also in Chinese. / Abstract --- p.I / 摘要 --- p.V / Acknowledgements --- p.VIII / Publications --- p.X / Table of contents --- p.XII / Abbreviations --- p.XV / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Allergic diseases and allergic inflammation --- p.1 / Chapter 1.1.1 --- Allergic diseases and their prevalence --- p.1 / Chapter 1.1.2 --- Immunopathology of allergic inflammation --- p.2 / Chapter 1.2 --- Biology of human eosinophils --- p.4 / Chapter 1.2.1 --- Morphology --- p.4 / Chapter 1.2.2 --- Cell surface receptors and mediators --- p.4 / Chapter 1.2.3 --- Origin and development of eosinophils --- p.7 / Chapter 1.3 --- Eosinophils and allergic inflammation --- p.7 / Chapter 1.3.1 --- Adhesion molecules on eosinophils for emigration --- p.8 / Chapter 1.3.2 --- Eosinophil activation and inflammatory mediators --- p.13 / Chapter 1.3.3 --- Survival of eosinophils in allergic inflammation --- p.18 / Chapter 1.3.4 --- Immunopathological roles of eosinophils in allergic inflammation --- p.18 / Chapter 1.4 --- Intracellular signaling mechanisms --- p.20 / Chapter 1.4.1 --- Signal transduction pathways of eosinophils --- p.20 / Chapter 1.4.2 --- Inhibitors of signaling molecules --- p.26 / Chapter 1.5 --- Aim of study --- p.29 / Chapter Chapter 2: --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Human eosinophils --- p.31 / Chapter 2.1.2 --- Cell culture --- p.33 / Chapter 2.1.3 --- Cell surface and intracellular immunofluorescent staining --- p.36 / Chapter 2.1.4 --- Detection of cytokine and chemokine release --- p.39 / Chapter 2.1.5 --- Detection of cell viability and apoptosis --- p.40 / Chapter 2.1.6 --- Protein extraction --- p.40 / Chapter 2.1.7 --- Western blot analysis --- p.40 / Chapter 2.1.8 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Purification of human eosinophils --- p.45 / Chapter 2.2.2 --- Cell culture --- p.46 / Chapter 2.2.3 --- Cell surface and intracellular immunofluorescent staining --- p.47 / Chapter 2.2.4 --- Detection of cytokine and chemokine release --- p.48 / Chapter 2.2.5 --- Detection of cell viability and apoptoas --- p.49 / Chapter 2.2.6 --- Protein extraction --- p.49 / Chapter 2.2.7 --- Western blot analysis --- p.50 / Chapter 2.2.8 --- SDS-PAGE --- p.50 / Chapter 2.2.9 --- Statistical analysis --- p.51 / Chapter Chapter 3: --- Role of Novel IL-1 Family Cytokine in Allergic Inflammation: IL-33-mediated Eosinophil Activation --- p.52 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Results --- p.54 / Chapter 3.2.1 --- "Total protein expression of IL-33 receptor, ST2, of human eosinophils" --- p.54 / Chapter 3.2.2 --- "Intracellular protein expression of IL-33 receptor, ST2，in human eosinophils" --- p.55 / Chapter 3.2.3 --- "Extracellular protein expression of IL-33 receptor, ST2, on human eosinophils" --- p.56 / Chapter 3.2.4 --- "Effects of IL-1β IL-18, and IL-33 on survival of human eosinophils" --- p.57 / Chapter 3.2.5 --- "Effects of IL-1β, IL-18, and DL-33 on surface adhesion molecule expression on human eosinophils" --- p.60 / Chapter 3.2.6 --- "Effects of IL-1β, IL-18, and IL-33 on chemokine and cytokine release from human eosinophils" --- p.64 / Chapter 3.2.7 --- "Synergistic effects of IL-1β, IH8, and IL-33 on IL-6 release from human eosinophils" --- p.69 / Chapter 3.2.8 --- "Effects of transcription and translation inhibitors on IL- 1β, IL-18, and IL-33-induced chemokine and cytokine release from human eosinophils" --- p.71 / Chapter 3.2.9 --- "Effects of different inhibitors on lL-1β, IL-18, and IL-33-induced survival enhancement of human eosinophils" --- p.75 / Chapter 3.2.10 --- Effects of different inhibitors on IL-1β and IL-33-mediated surface expression of adhesion molecules on human eosinophils --- p.77 / Chapter 3.2.11 --- "Effects of different inhibitors on IL-33, IL-1β，and IL-18-induced release of CCL2，CXCL8，and IL-6 from human eosinophils" --- p.79 / Chapter 3.2.12 --- "Effects of IL-33, IL-1β and IL-18 on activation of ERK, p38 MAPK, and NF-kB pathways in human eosinophils" --- p.83 / Chapter 3.3 --- Discussion --- p.85 / Chapter Chapter 4: --- Co-culture of Eosinophils & Epidermal Keratinocytes Upon IL-33 Stimulation: Implications for Immunopathology of Atopic Dermatitis --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Results --- p.98 / Chapter 4.2.1 --- Effect of IL-33 on surface expression of CD18 and ICAM-1 upon the interaction of human eosinophils and epidermal keratinocytes --- p.98 / Chapter 4.2.2 --- Effect of IL-33 on CCL2 release upon the interaction of human eosinophils and epidermal keratinocytes --- p.98 / Chapter 4.2.3 --- Effect of IL-33 on CXCL8 release upon the interaction of human eosinophils and epidermal keratinocytes --- p.101 / Chapter 4.2.4 --- Effect of IL-33 on IL-6 release upon the interaction of human eosinophils and epidermal keratinocytes --- p.101 / Chapter 4.2.5 --- Source(s) of CCL2 in co-culture of human eosinophils and epidermal keratinocytes upon IL-33 stimulation --- p.104 / Chapter 4.2.6 --- Source(s) of CXCL8 in co-culture of human eosinophils and epidermal keratinocytes upon IL-33 stimulation --- p.105 / Chapter 4.2.7 --- Source(s) of IL-6 in co-culture of human eosinophils and epidermal keratinocytes upon BL-33 stimulation --- p.108 / Chapter 4.2.8 --- "Effect of transwell insert on the induction of CCL2，CXCL8, and IL-6 release in co-culture of human eosinophils and epidermal keratinocytes upon IL-33 stimulation" --- p.110 / Chapter 4.3 --- Discussion --- p.113 / Chapter Chapter 5: --- Concluding Remarks and Future Perspectives --- p.120 / Chapter 5.1 --- Concluding Remarks --- p.120 / Chapter 5.2 --- Future Perspectives --- p.123 / Appendix --- p.126 / References --- p.127

Indiana University-Purdue University Indianapolis (IUPUI) / Damage Associated Molecular Patterns (DAMPs) act largely as endogenous ligands to initiate and maintain the signaling of both inflammatory processes and the acquired immune response. Prolonged action of these endogenous signals are thought to play a significant role sterile inflammation which may be integral to the development of chronic inflammation pathology. HMGB1 (High Mobility Group Box 1) is a highly conserved non-acetylated protein which is among the most important chromatin proteins and serves to organize DNA and regulate transcription. Following stress or injury to the cell, hyperacetylation of lysine residues causes translocation of HMGB1 and eventual release into the extracellular environment where it can take the form of a DAMP and interact with cell types bearing either the Receptor for Advanced Glycation End-products (RAGE) or Toll-Like Receptor 4 (TLR4). Activation of these surface receptors contribute directly to both acute and chronic inflammation. This project investigated the role of HMGB1 through its receptors Receptor for Advanced Glycation End-products (RAGE) and Toll-Like Receptor 4 (TLR4) as it pertained to the development of chronic inflammation and pathology in small diameter, nociceptive sensory neurons. It was demonstrated that the neuronal signaling associated with exposure to HMGB1 is dependent upon the ligands conformational states, as the state dictates its affinity and types of neuronal response. Neuronal activation by bacterial endotoxin or the disulfide state of HMGB1 is dependent on TLR4 and the associated signaling adapter protein, Myeloid differentiation primary response gene 88 (MYD88). Interruption of the receptor-mediated signaling cascade associated with MyD88 was shown to be sufficient to mitigate ligand-dependent neuronal activation and demonstrated significant behavioral findings. Further downstream signaling of HMGB1 in the neuron has yet to be identified, however important steps have been taken to elucidate the role of chronic neuroinflammation with hopes of eventual translational adaptation for clinical therapeutic modalities.

La voie des kynurénines est l’une des trois voies de biosynthèse du NAD, qu’elle produit à partir de la dégradation du plus rare des acides aminés essentiels, le tryptophane. Cette voie catabolique est initiée par trois enzymes distinctes, dont l’indoléamine 2,3-dioxygénase 1 (IDO-1). L’IDO-1 est la seule des trois enzymes dont l’expression est induite par des stimuli pro-inflammatoires tels que l’IFN-γ, le TNF-α ainsi que des produits bactériens et viraux. De plus, son expression est induite de manière particulièrement importante au cours de la maturation des cellules dendritiques (DC) qui jouent un rôle clé, tant dans les réponses immunes innées qu’adaptatives. Par conséquent, outre la production d’un cofacteur essentiel du métabolisme cellulaire, le catabolisme du tryptophane participe également à la régulation de la réponse immune. <p><p>En effet, la fonction d’agent antimicrobien fut la première attribuée à l’IDO-1 parce qu’en réponse aux stimuli inflammatoires, celle-ci dégrade le tryptophane et limite la prolifération d’organismes pathogènes auxotrophes pour cet acide aminé. Ensuite, l’inhibition de l’activité catalytique de l’IDO-1 par une approche pharmacologique permit de mettre en évidence la contribution de cette enzyme au phénomène de tolérance maternelle pour les fœtus semi-allogéniques qu’elle porte. Enfin, sa participation à la tolérance périphérique fut proposée étant donné sa capacité à induire le développement de Treg ou à leur servir de mécanisme effecteur. Cependant, ces deux dernières fonctions ne sont pas très claires puisque les souris invalidées pour le gène de l’IDO-1 (IDO-1-/-) ne présentent ni défaut reproducteur, ni signe d’auto-immunité spontanée.<p><p>Comme deux publications récentes suggéraient que l’inhibition pharmacologique de l’IDO-1 affecte la maturation des moDC humaines, nous avons effectué une analyse détaillée du compartiment immunitaire des souris IDO-1-/-, avec une attention toute particulière pour les DC. Au début, nous avons également constaté un important défaut de la maturation des BMDC IDO-1-/- générées in vitro en présence de GM-CSF. Cependant, le défaut se révéla extrêmement dépendant des conditions de culture, puisqu’un changement de substrat de culture ou de facteur de croissance suffit à restaurer une maturation normale de ces cellules. De même, l’analyse de la maturation des DC spléniques démontra de manière claire que l’IDO-1 n’est certainement pas essentielle à la maturation des DC in vivo.<p> <p>Nous avons ensuite montré que l’expression fonctionnelle d’IDO-1 protège les cellules et potentiellement les souris qui l’expriment lorsqu’elles sont soumises à des stress oxydatifs, suggérant que l’IDO-1 puisse consommer les anions O2- afin d’assurer son activité catalytique. Contre toute attente, nous avons ensuite constaté que les souris IDO-1-/- survivent plus longtemps aux infections par la forme pléiomorphe du parasite Trypanosoma brucei brucei. Bien que la levée de l’inhibition de la lymphoprolifération chez les souris IDO-1-/- soit l’explication la plus évidente de l’augmentation de leur survie, nous suggérons plutôt que c’est la perte de la fonction antioxydante de l’IDO-1-/- qui leur confère cette résistance. <p><p>En conclusion, l’IDO-1 ne semble pas jouer un rôle important dans la différenciation et maturation des cellules dendritiques. Nos observations préliminiares indiquent cependant que cette enzyme pourrait jouer un rôle anti-oxydant, et protége donc les cellules dendritiques d’un stress oxydant potentiellement causé lors des réponses innées anti-microbiennes.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Throughout this work, we looked at P2X coupled pathways in macrophages. We worked on three different models of macrophages in order to establish the best model to understand the role of P2X4 receptors in the inflammation. Our work also consisted of further characterizing P2X7 receptor dependent pathways in these models. <p>P2X4 and P2X7 receptors are ionotropic receptors which are expressed by a variety of immune cells including macrophages. Macrophages play a very important host defense function as they are major actors in the innate immune system and they can initiate the activation of the adaptive immune system. The endogenous ligand of P2X receptors is ATP for which they share very different sensitivities. P2X4 receptors are relatively sensitive to this agonist while P2X7 receptors require concentrations > 100 μM ATP to be activated. <p>Our study supports the expression of P2X4 and P2X7 receptors in J774.2 murine macrophages and in human macrophages. Additionally, we worked on murine peritoneal macrophages for which the existence of P2X4 and P2X7 receptor expression had previously been shown in our lab. <p>A wide range of different macrophage phenotypes exist. Two extremes determine an array of phenotypes which are delimited by M1 pro-inflammatory macrophages and M2 anti-inflammatory macrophages while Mφ macrophages define the center of the array. Most of the work exposed in this study was carried out on pro-inflammatory macrophages which were obtained either by priming the cells with LPS alone (Mφ + LPS) or by polarizing them with LPS in association with IFNγ (M1). <p>We show in this study that LPS-primed J774.2 murine macrophages are not a good model to study the role of surface P2X4 receptor in pro-inflammatory macrophages. Additionally, we support that murine peritoneal macrophages primed with LPS are a good model to understand the hypothetical role of P2X4 receptors in the inflammation. Finally, we suggest that human M1 macrophages could be as well. Next, we also confirm that J774.2 murine macrophages, murine peritoneal macrophages and human macrophage express functional P2X7 receptors. In this study, we show that P2X7 receptors are coupled to RONS formation in J774.2 murine macrophages and to AA release through PLA2 activation in peritoneal macrophages. We show that activation of J774.2 murine macrophages with high concentrations of ATP (>600 μM) stimulates ROS formation including mitochondrial superoxide anions. In addition, our work shows the importance in using different dyes and suggests that different types of ROS play different functions in P2X7 receptors downstream pathways. <p>Next, we show that P2X7 receptor activation is coupled to an iPLA2 activity and that the release of free fatty acids mediated by 1 mM ATP is p-ERK1/2 dependent in LPS-primed murine peritoneal macrophages. <p>In addition, we have evaluated the effect of hypoxia on pathways which have been reported to be coupled to P2X7 receptor activation in pro-inflammatory human macrophages. Hypoxia does not seem to modulate P2X7 receptor functionality. However, both acute and chronic hypoxia influenced P2X7 receptors downstream pro-inflammatory coupled pathways. Finally, our work has enabled us to suggest for the first time that IFNγ plays an important function in host defense mediated by human P2X7 receptor activation in a hypoxic environment. <p>The effect of extracellular environment and thus different macrophage phenotypes have also been evaluated throughout this work in which we looked at the effect of polarization on P2X4 and P2X7 receptor functionality. Our work shows that LPS-priming does not modulate P2X4 receptor functionality in murine macrophages. Next, through our work, we suggest that polarization of human macrophages affects P2X4 receptor functionality in human macrophages. Additionally, our work shows that LPS affects ATP-mediated RONS formation in J774.2 murine macrophages but not P2X7-mediated AA release in primary murine macrophages. <p><p>Overall, first, our work has enabled us to suggest macrophage models to use in order to study the hypothetical role of P2X4 receptor in the inflammation mediated by macrophages. Second, it has allowed us to further understand how P2X7 receptors can act as important mediators of the immune system mediated by macrophages. In addition, many interesting observations were made in this study which allows us to propose several options for future directions. To finish, our work underlines the importance of the extracellular environment in some pathways mediated by ATP in macrophages.<p> / Doctorat en sciences pharmaceutiques / info:eu-repo/semantics/nonPublished

Sickle cell disease is a hereditary blood disorder caused by a point mutation in the gene encoding hemoglobin. This mutation causes hemoglobin molecules to polymerize during de-oxygenation of erythrocytes producing rod-shaped polymers that bend and distort the red blood cell membrane, making it more rigid and “sickled”. This sickling causes red blood cells to lose their flexibility and ability to navigate small capillaries and also enhances the production of pro-inflammatory membrane-derived microparticles, leading to chronic inflammation and many complications such as peripheral artery disease, stroke, myocardial infarction, vasculitis and even death. Sphingolipids are a class of lipids containing a backbone of sphingoid bases and are integral components of erythrocyte and microparticle membranes. Many of these lipids are known to mediate biological processes, but their expression, distribution and orientation in erythrocytes during sickle cell disease has never been explored. Sphingomyelin, the most abundant sphingolipid in the red blood cell membrane is hydrolyzed by sphingomyelinase to produce ceramide, which has been shown to alter membrane dynamics and enhance microvessel formation. Additionally, ceramide can be further metabolized to form sphingosine and sphingosine 1-phosphate, which is a bioactive ligand for 5 known G-protein coupled receptors present on most blood and vascular cells that modulates cell motility, proliferation, migration and phenotype. Prior to this work, it was not understood how sphingolipid metabolism contributes to vascular inflammation in sickle cell disease. Together, this body of work has elucidated key enzymatic and lipidomic alterations in sphingolipid metabolism (i.e. the activation of acid sphingomyelinase on red blood cells) that result in the production of sphingolipid-rich erythrocyte-derived microparticles, which enhance inflammatory cell activation. Our work has elucidated novel pharmacological targets to reduce microparticle generation and subsequent vascular inflammation in sickle cell disease.

Motivation In South Africa, various transitional changes parallel detrimental modifications in lifestyle behaviour of especially the lower socio-economic communities. We are currently double-burdened by a high prevalence of communicable and noncommunicable diseases such as diabetes, chronic respiratory and cardiovascular diseases, which is accompanied by a high cardiovascular mortality rate. Healthcare and treatment resources are limited and low-cost intervention strategies to lower this burden are urgently needed. Unhealthy lifestyle behaviours, such as excessive alcohol consumption and tobacco use, are known to augment inflammation as reflected by inflammatory markers such as C-reactive protein and interleukin-6, which are well-known risk factors for cardiovascular disease and mortality. Several studies showed the prognostic value of soluble urokinase plasminogen activator receptor (suPAR) in advanced disease states and that suPAR associates with different types of cancers, infectious diseases, diabetes, coronary artery disease and all-cause mortality. Since the discovery of suPAR in 1991, the role of this less known inflammatory marker in various diseases has been under debate. It was further reported that black individuals have higher suPAR levels than whites. However, whether an unhealthy lifestyle and cardiometabolic risk factors are related to suPAR, whether suPAR plays a role in the development of cardiovascular disease such as hypertension, and whether suPAR could predict all-cause and cardiovascular mortality, especially among the understudied black South African population, remain to be established. Aim The central aim of this thesis was to determine if suPAR associates with cardiovascular disease development in a black South African population. We therefore explored whether suPAR relates to lifestyle and cardiometabolic risk factors, associates with the development of hypertension and has prognostic value for cardiovascular and all-cause mortality over five years. Methodology This five-year prospective sub-study, which is embedded in the international Prospective Urban and Rural Epidemiology study, included black South African volunteers of ages older than 35 years from the North West province, South Africa. Baseline data collection took place in 2005 during which 2 010 men and women from urban and rural areas were examined. A total of 1 292 participants returned for examination and were followed-up for the first time in 2010. Of these participants, 214 were newly identified as being infected with the human immunodeficiency virus (HIV), while 233 died during the five year period. Standardised methods were used to capture all data and included health questionnaires (lifestyle factors, medication use, disease status and history, mortality outcome), cardiovascular and anthropometric measurements, as well as biochemical analyses of inflammatory markers (suPAR, C-reactive protein, interleukin-6), HIV status and relevant metabolic markers. In preparation for statistical analyses, non-Gaussian variables were logarithmically transformed. We compared means and proportions with independent t-tests, analysis of variance, analysis of covariance (for adjustments) and Chi-square tests, while dependent t-tests and McNemar tests were used for analysis of longitudinal data within individual groups. We determined relationships between variables with Pearson’s correlation coefficients. Independent relationships were determined with logistic regression, forward stepwise multiple regression and proportional Cox-regression analyses. Mortality rates were calculated using Kaplan-Meier survival function estimates and log-rank tests. In all cases, p≤0.05 were used to indicate statistical significance. Results and conclusions of each manuscript Three manuscripts were written in order to achieve the aim of this thesis. In the first manuscript we explored the cross-sectional relationships of suPAR with lifestyle and cardiometabolic risk factors in a black South African population. We showed that suPAR was independently associated with lifestyle behaviours, including alcohol consumption, as indicated by gamma-glutamyltransferase levels (β=0.24; p<0.001), tobacco use (β=0.13; p<0.001) and unemployment (β=0.07; p=0.039), despite no direct links with cardiometabolic factors such as blood pressure, dyslipidaemia, glycaemia or adiposity. These findings emphasise the important need to address lifestyle behaviours in order to limit the detrimental effect of modifiable risk factors on the health and mortality rate of this population. Secondly, we determined whether suPAR was associated with the development of hypertension over five years. We found that suPAR was higher and increased more prominently (14.2% vs. 6.94%; p=0.007) in participants that developed hypertensio than in those that remained normotensive. Change in systolic blood pressure was independently associated with baseline suPAR (β=0.14; p=0.043), while becoming hypertensive was associated with an increase in suPAR (odds ratio=1.41; p=0.015). Whether inflammation leads to the development of hypertension or vice versa, remains unclear. Our findings emphasise the need to acknowledge the role of inflammation in hypertension and may permit further investigation of the use of suPAR as a potential marker for early risk identification and intervention. The third manuscript investigated the prognostic value of suPAR, compared to other inflammatory markers C-reactive protein and interleukin-6, in all-cause and cardiovascular mortality. We showed for the first time in a black population that suPAR predicted both all-cause (hazard ratio=1.27; p=0.003) and cardiovascular mortality (hazard ratio=1.40; p=0.026), independent of interleukin-6. Future research is needed to clarify the mechanisms behind the association of suPAR with cardiovascular mortality and to explore the possibility of a suPAR cut-off value for early identification of those with increased risk for cardiovascular morbidity and mortality in this population. General conclusion In this thesis we showed for the first time that suPAR has potential as a marker of cardiovascular disease development in black South Africans. SuPAR associated with hypertension and independently predicted all-cause and cardiovascular mortality over five years. Our findings, that suPAR is independently associated with adverse health behaviours such as alcohol and tobacco use, lend support for the use of suPAR as a novel approach for early risk identification and intervention strategies, which may be effective in combatting the high cardiovascular disease burden among the black South African community. / PhD (Physiology), North-West University, Potchefstroom Campus, 2015