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Eosinophils are characteristic infiltrating cells in asthmatic airways and may contribute to allergic and inflammatory diseases pathology through the ability of their products to produce chronic inflammatory and remodelling processes. One focus of efforts to define new therapeutic targets for chronic inflammatory diseases has been the study of mechanisms through which eosinophils are recruited to tissues and become activated, leading to secretion of reactive oxygen species, inflammatory mediators, cationic proteins and cytokines. The signalling pathways responsible for evoking migratory and secretory response in human eosinophils are incompletely understood.

Hypothesis: ECMO is an acceptable supportive therapy for patients with severe SIRS despite triggering haematic response.;Objectives: To develop a reliable and reproducible animal model of SIRS to investigate strategies for reducing the haematic response.;Materials and Methods: Literature review: (I) The Systemic Thrombo-inflammatory Pathway (STIP). (II) The haematic response. Clinical studies: (I) Institutional review of ECMO for severe SIRS. (II) Comparative review of oxygenators performance. In-vivo studies: Animal experiments, (I) Development of a rabbit model of graded SIRS. (II) Dose response relationship between administered intravenous endotoxin and fatal Multi-organ Dysfunction Syndrome (MODS). (III) Inter-individual variation amongst rabbits receiving a lethal dose of endotoxin. In-vitro studies: Evaluation of the cellular and biochemical components of STIP in rabbits. (I) Development of rabbit ELISA. (II) Determination of the normal range, dose response and inter-individual variation. (III) Immunohistochemical evaluation of endotoxin induced lung injury. (IV) Dose related oxidative stress and DNA damage and (V) apoptosis in rabbit lung.;Results: There is a reciprocal relationship between graded SIRS and the outcome of ECMO. There is a linear relationship between the dose of endotoxin and the development of graded SIRS in rabbits. Significant DNA homology and cross-reactivity exists between humans and rabbits making this a useful model for immune experimentation.;Conclusions: ECMO is superior to conventional ICU management in selected groups of patients. New oxygenator technology has significantly reduced the haematic response to ECMO, however it has failed to influence survival. Cellular components such as neutrophils play a central role in SIRS activation, however thrombin appears to be the common biochemical component for feedback escalation and progression of severe SIRS to MODS.

The aims of the work described in this thesis are to examine the impact of physical activity/exercise and energy balance on metabolic control and inflammation and specifically whether exercise has independent benefits on various health-related outcomes above a role in energy balance. Chapter 3 examined whether a lifestyle intervention combining dietary advice with increased physical activity would further improve inflammatory markers compared to dietary advice alone and usual care in 494 patients with newly diagnosed type 2 diabetes. Motivational unsupervised diet and diet plus physical activity interventions led to reductions in inflammatory markers. Interestingly, there was no greater benefit from adding physical activity advice to dietary advice. Chapter 4 investigated whether daily vigorous-intensity exercise would counteract the metabolic changes induced by short-term overfeeding and reduced physical activity independent of any net attenuation of energy imbalance in healthy young men. The overfeeding and reduced activity model induced a state of insulin resistance, hyperinsulinaemia and altered expression of several key genes within adipose tissue. The inclusion of a daily vigorous-intensity exercise bout largely prevented these changes from taking place independent of any net effect on energy imbalance. Chapter 5 examined whether caloric restriction combined with vigorous-intensity exercise would further improve metabolic control and inflammatory markers compared to moderate-intensity exercise in middle-aged, overweight/obese men and postmenopausal women. Three weeks of caloric restriction combined with either vigorous or moderate-intensity physical exercise improved insulin sensitivity, lipid profiles and markers of inflammation. These results confirm the positive effects of combined caloric restriction and increased exercise in sedentary overweight men and women, but that exercise intensity does not seem to be so important. In conclusion, this thesis presents reasonable evidence that exercise per se has a positive impact upon metabolic control and inflammation independent of energy balance during an energy surplus but that a role in contributing to the health benefits during an energy deficit are less convincing.

My research has strived to understand two different aspects of immune cell response to stimuli: i) cross talk between innate and adaptive immune responses; ii) regulation of inflammatory signalling by a novel phosphoprotein, Arhgef101. Canonical and non-canonical NFκB signalling pathways have been described to date, each inducing a distinct gene expression profile. Current understanding indicates the canonical being mainly involved in innate- and non-canonical being mostly involved in adaptive immunity. Using a T cell hybridoma model, I established that chronic activation of the canonical pathway by TNFα led to up-regulation of components of the non-canonical pathway, specifically Re1B and p100. Hypothesising that cells exposed chronically to TNFa would then have enhanced non-canonical activation propagating the inflammatory response, CD27 was identified as a model system for activating the non-canonical NFκB pathway. In spite of exhaustive attempts with this system and other receptor systems, none were found to be capable of inducing robust and reproducible non-canonical NFκB activation. We have identified a novel phosphorylation designated Arhgef101. Arhgef101 was found to undergo tyrosine phosphorylation in the murine macrophage cell line, RAW 264.1, after LPS stimulation. Murine tissue screening for Arhgef101 RNA showed ubiquitous expression. No compartmental, membrane or actin filament associations were observed. Overexpression of Arhgef101 in RAW cells resulted in overproduction of TNFa but not IL-6 after TLR4 stimulation, when compared to untransfected cells. Overexpression in the murine fibroblast cell line, NIH 3T3, amplified both IL6 and KC production in response to IL1 and LPS. Primary-transcript quantitative PCR suggests a potential transcriptional regulatory role for Arhgef101. With no detectable differences in the major signalling pathway NFκB, ERK, JNK, or p38 MAPK these data implicate Arhgef101 as a potential signalling adaptor involved in an, as yet, unidentified signalling pathway.

The systemic inflammatory response (SIRS), sepsis and septic shock are termed collectively the septic syndromes and are leading causes of morbidity and mortality amongst patients admitted to the intensive care unit (ICU). The pathophysiology of the syndromes is consequent upon the interaction between the infecting agent (or insult) and the host immune response. Persistence of the immune response and continued oxidative stress are thought to be key components. The purpose of this project was to investigate the implications of HO-1 expression on the inflammatory response, using in vivo, in vitro and ex vivo models.