Inflammation involves interactions amongst many different cell types as a defense mechanism of the body. Inflammation is also involved in cardiovascular disease (CVD). The role of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs) in modulating the inflammatory response has been proposed. The aim of these studies is to investigate the effects of modest intakes of n-3 PUFAs on CVD risk factors especially inflammatory markers, including soluble adhesion molecules, in adult humans with and without CVD and to identify the effects of selected fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on inflammatory responses, especially adhesion molecule expression in cultured human endothelial cells of different origin (fetal vs. adults; vein vs. artery). In the first in vivo study, healthy middle-aged men aged 35-60 years were randomized to 1.8 g/d EPA plus 0.23 g/d DHA (n = 9) or placebo oil (2.6 g/day medium-chain saturated fatty acids; n = 11) for 8 weeks. In a second in vivo study, patients awaiting carotid endarterectomy were randomised to 0.8 g/d EPA plus 0.67 g/d DHA (Omacor; n = 47) or olive oil (n = 53) as placebo for between 7 and 102 days until surgery. Supplementation with fish oil in healthy men resulted in a 363% increase in EPA and only a 13% increase in DHA in plasma phosphatidylcholine (PC). On the other hand, Omacor supplementation resulted in significantly increased EPA and DHA in plasma PC by 161% and 70%, respectively. In healthy subjects, there was very little effect of n-3 fatty acids on the risk factors measured (lipid profiles and inflammatory markers), apart from a reduction in plasma soluble intercellular molecule-1 (sICAM-1) concentration compared with placebo (P = 0.05). The change in plasma sICAM-1 concentration was significantly inversely associated with the change in DHA in plasma PC (r = -0.675; P = 0.001). Supplementation with Omacor, however, significantly decreased total plasma cholesterol, triacylglycerol (TAG) and LDL-cholesterol concentrations (P < 0.001) by 13%, 14%, and 5% respectively. In terms of inflammatory markers, supplementation with Omacor significantly decreased sE-selectin by 23% (P = 0.006) and sVCAM-1 by 25% (P < 0.0001), and had no significant effects on other plasma inflammatory markers including sICAM-1 even though trends toward decreases in these markers were observed. This study suggests some anti-inflammatory actions of moderate dose of Omacor in carotid endarterectomy patients. Based on correlation analysis between mRNA expression of inflammatory markers in plaque and plasma concentrations, it seems that soluble inflammatory markers cannot be used to reflect the expression of these molecules at the cell surface, i.e. in the vasculature or in the plaque. In the in vitro experiments the inflammatory stimulus lipopolysaccharide (LPS) up-regulated all three adhesion molecules studied at the protein (as assessed by ELISA) and the mRNA (as assessed by reverse transcription and real-time PCR) levels. VCAM-1 was affected by fatty acids to a greater extent than ICAM-1 or E-selectin. Amongst the fatty acids, DHA has the greatest and the most consistent effects on adhesion molecule protein expression. EPA was also a potent fatty acid inhibitor of adhesion molecule expression at the mRNA level. Some effects of stearic, oleic and arachidonic acids on adhesion molecules were also seen. The effects of fatty acids on the adhesion molecule expression were fatty acid, adhesion molecule and endothelial cell specific. The inhibitory effects of fatty acids were more pronounced in vein endothelial cells than arterial endothelial cells. The precise underlying mechanism on how fatty acids affect adhesion molecule expression remains to be clarified.

Autoimmune diseases (AID) are chronic inflammatory diseases (CID) mediated by selfreactive T and B cells and are generally the results of the breakdown of T cell tolerance to self-antigen and failure of peripheral regulatory mechanisms. In this thesis I studied different mechanisms controlling the development of CIDs. I investigated the initial events involved in the activation of self-reactive CD4+ T cells which mediate the destruction of the thyroid in a mouse model of spontaneous thyroiditis. TAZ10 transgenic mice express a human T cell receptor (TCR) specific for a cryptic epitope of thyroid peroxidise (TPO) generated upon endogenous processing by thyroid epithelial cells (TEC), and a naturally occurring antagonistic epitope presented by dendritic cells (DC) upon exogenous processing of TPO. I have characterized the function of myeloid derived suppressor cells (MDSCs) in TAZ10 mice. MDSCs accumulate in lymphoid and non-lymphoid organs of TAZ10 mice during acute phases of inflammation and their number decrease as inflammation is fading. Despite their strong inhibitory function on T cell function and proliferation, MDSCs fail to prevent the activation of self-reactive T cells. I showed that the manipulation of MDSCs generated DCs that efficiently promoted the activation of T cells from TAZ10 mice. By contrast, peripheral T cells from patients with rheumatoid arthritis (RA) and lupus had a high proliferative activity compared to controls. Further analysis revealed that RA patients had reduced amounts of inhibitory MDSCs in peripheral blood. I showed that in TAZ10 mice TEC upregulate MHC class II molecules and present the cryptic epitope to TAZ10 T cells inducing their activation. I have demonstrated that DCs are responsible for the spreading of the TPO cryptic epitope from the thyroid to draining lymphnodes (DLN) resulting in the strong activation of transgenic T cells from TAZ10 mice. By adoptive transfer experiments, I showed that the activation of naive TAZ10 T cells occurs within days both in the thyroid and draining lymph-nodes (DLN) and resulted in the destruction of the thyroid. Altogether, this work shows for the first time that in a model devoid of any environmental insults, the normal turnover of TEC is sufficient to induce the activation of self-reactive T cells and the development of AID. In this thesis, I have highlighted the potential role of tissue transglutaminse 2 (TG2) in the treatment of CIDs. TG2 contributes to the pathogenesis of celiac disease and I have showed that TG2 activity promotes inflammation in patients with cystic fibrosis (CF). Mutation of the cystic fibrosis transmembrane regulator gene (CFTR) in CF patients is associated with increased TG2 expression and activity. In CF, TG2 promoted the crosslinking of the antiinflammatory peroxisome proliferator-activated receptor (PPAR) into perinuclear agresomes. The functional sequestration of PPAR was leading to increased inflammation. The finding of this function of TG2 in CF was relevant in TAZ10 mice as in-vivo inhibition of TG2 downregulated common markers of inflammation.

No description available.

Chronic obstructive pulmonary disease (COPD) is a debilitating disease that as of yet has no cure and therapy is limited to symptomatic relief. A major risk factor for the development of COPD is smoking, although the disease does have some component of genetic predisposition. In excess of £500 million funding per year is required to accommodate the needs of COPD patients and approximately 27, 000 deaths per year in the UK can be attributed to COPD. COPD is comprised of three conditions-chronic bronchitis, bronchiolitis and emphysema and these will be present in the COPD patient to varying degrees. It is well accepted that COPD is a disease characterised by increases in inflammation and as such inflammatory stimuli, such as cigarette smoke and lipopolysaccharide (LPS) from bacterial cell walls, have been associated with disease development and progression. Consequently, there are associated increases in inflammatory cytokines such as TNFα in the COPD patient. Although inflammation plays a major role in the pathogenesis of COPD there are other factors to consider such as proteolytic damage caused by disturbances in the proteinase/anti-proteinase balance. This is of particular importance in emphysema where increases in neutrophil elastase concentration results in the destruction of elastin fibres which results in a decrease in lung function. Elastase is also known to contribute to the mucus hypersecretion associated with chronic bronchitis. The proteolytic actions of elastase are well characterised but there is gathering evidence to suggest that it may also be able to act as an inflammatory stimulus, thereby increasing its role in the pathogenesis of COPD. This study has utilised a human lung explant model to investigate whether elastase can initiate an inflammatory response; concentrations of the pro-inflammatory cytokine TNFα and the anti-inflammatory cytokine IL-10 in the culture supernatant have been investigated as part of this. Data from this model (n=36) has shown that elastase can significantly increase both TNFα and IL-10 compared to control. Elastase stimulation, for 24hrs, caused the release of 30.1±8.0pg TNFα/mg tissue and 3.1±0.5pg IL-10/mg tissue. This response is comparable to that produced by LPS. We have also found that elastase can induce a Th2 type response from the parenchymal explants, with increases in IL-4, IL-5 and IL-13. The inflammatory response detailed in this study appears to be unique to elastase and cannot be reproduced with other serine proteinases, such as trypsin and chymotrypsin, or a cysteine proteinase, papain. Our data has also shown that the proteolytic activity of elastase can be inhibited by an elastase-specific inhibitor, elastatinal, and by doing so attenuates the TNFα response; elastase stimulation alone produced 127.5±72.1pg/mg tissue, whereas with the inhibitor this production dropped to 40.4±9.0pg/mg tissue. As of yet, the exact mechanism by which elastase induces inflammation is unknown but we have investigated the relationship between elastase and two candidate receptors-proteinase-activated receptor (PAR)-2 and Toll-like receptor (TLR)-4. Although elastase stimulation does not appear to alter the gross amount of these receptors present in the parenchyma, we have found that those patients with mild to moderate COPD tend to have greater levels of both PAR-2 and TLR-4. We have also utilised synthetic activating peptides for PAR-2 and in comparison to elastase stimulation it is suggested that elastase may cause its inflammatory and Th2 effects via distinct pathways.