Small heat-shock proteins are conserved molecular entities present in all mammalian cells. They have historically been studied in the context of being intracellular molecular chaperones that are constitutively expressed, with a capacity to be induced by cellular stress, in order to promote and remediate protein folding. More recently however, growing evidence suggests that the action of small heat-shock proteins is not limited to protein folding but also extends to a wider range of important cellular roles. Of the 11 small heat-shock proteins that are expressed in mammalian cells, only 4, HSPB1, HSPB5, HSPB6 and HSPB8, are expressed in the central nervous system (CNS). The role of these small heat-shock proteins in the CNS is thought to be protective as they show widespread upregulation during several neurological conditions. Confoundingly however, studies from R6/2 animal models of Huntington’s disease show a selective reduced expression of HSPB5 in these animals, raising pertinent questions as to whether this reduction is cause or effect of the condition. Here, we have investigated whether reduced expression of HSPB5 has a detrimental effect, focussing specifically on HSPB5’s proposed immune modulatory role. Using mice inoculated with S. typhimurium, we found that, in our hands, mice lacking HSPB5 did not appear to be phenotypically different from wild type animals and equally, the reduced expression of HSPB5 did not exacerbate systemic inflammation or potentiate disease progression. Furthermore, to investigate HSPB5’s role in the CNS, we inoculated animals with ME7 Prion and also found that deficiency in HSPB5 did not alter phenotype or behaviour and did not negatively influence disease progression. Lastly, we investigated whether the reduced expression of HSPB5 as observed in R6/2 animals was reciprocated in humans. Our findings show that in humans disease, there is no reduction of HSPB5. Our findings suggests that in C57BL/6 animals, HSPB5 does not appear to have an immune modulatory role; they also highlight how data obtained from animal models should be taken tentatively.

The bacterial pathogen Chlamydia trachomatis causes Trachoma, the worlds leading cause of preventable blindness and is also responsible for the most common curable sexually transmitted disease in the UK and United States. C. trachomatis is an obligate intracellular organism characterised by a unique and complex growth cycle. Its study presents many challenges since it has historically been recalcitrant to genetic manipulation and growth in the absence of a host cell. Nevertheless, the sequencing of the C. trachomatis genome and its relatively small size by comparison to genomes from other bacterial pathogens, has paved the way for studies at the proteomic level. This thesis describes the development and application of proteomic approaches to study C. trachomatis L2. To survey the expressed chlamydial proteome, a combination of the qualitative approaches, 2-DGE, MudPIT and GeLC-MS/MS; and the quantitative approaches AQUA, iTRAQ and LC-MSE were used. Collectively, the approaches efficiently identified 648 expressed proteins, representing ~72% of the predicted proteome of C. trachomatis L2, from both the infectious (elementary body, EB) and replicating (reticulate body, RB) form of the pathogen. In the infectious EB, the entire set of predicted glycolytic enzymes were detected, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. Further, proteomic analysis of the RB form also uncovered biosynthetic enzymes for chlamydial cell wall synthesis, indicating that peptidoglycan is produced in some form during growth in host cells. Comparison of the quantitative approaches iTRAQ and LC-MSE demonstrated that LC-MSE quantitative data was significantly more robust and extensive relative to iTRAQ data. In addition to information on relative amounts of these proteins between the two forms, LC-MSE data also yielded the cellular concentration (molecules per cell) for 489 proteins. This extensive set of absolute quantitation data permits estimates of the energy invested in the synthesis of various classes of proteins. The results indicate that C. trachomatis devotes most of its energy into maintenance of the translational machinery. However, it also expends significant amounts of energy into making cell envelope components and a set of hitherto hypothetical proteins. These proteins, which account for the bulk of the energy invested by the intracellular RB form of the pathogen as it converts to the extracellular EB form, highlight the importance of absolute quantitation data for understanding the biological processing status of the cell. The datasets also revealed a large number of proteins that were differentially expressed between replicating RBs and infectious EBs, ranging from 8.4-fold down-regulation to 3.5-fold up-regulation. Consistent with transcriptomic studies (Belland et al., 2003), proteins involved in protein synthesis, ATP generation, central metabolism, secretion and nutrient uptake were predominant in the metabolically active RB at 15 h PI. Although many of the proteins in these functional categories were down-regulated in EBs, proteins required for glycolysis, central metabolism, protein synthesis, and type III secretion were present in significant amounts in EBs suggesting that the infectious EB is primed ‘ready-to-go’ upon contact with the host cell.

No description available.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the developed world, but the mechanisms leading to AMD are poorly understood. Circulating retinal autoantibodies and antibody deposits in the retina are associated with AMD but despite this relationship, immune complex (IC)-mediated responses and underlying mechanisms of inflammation in the retina have not been characterised. IgG antibodies can activate immune effector function through formation of IC and their interaction with Fcγ receptors (FcγR) expressed by immune cells. This study aims to test the hypothesis that IC formed in the retina induce an inflammatory response following interaction with activating FcγRs expressed on microglia and/or macrophages, which may contribute to the pathogenesis of AMD. To study the biological effect of IC formation in the retina a model of IC injury was developed and fully characterised. The involvement of mouse FcγRs (mFcγRs) was first studied using Fc gamma chain deficient (γ-/-) mice, lacking cellular expression of activating mFcγRs, and further characterised using FcγRI-/-, FcγRIII-/- and FcγRIV-/- mice. The presence of IC and human FcγR (hFcγR) expression was investigated in human donor eyes from early and wet AMD patients and healthy controls. Finally the effect of inflammatory mediators on human retinal pigmented epithelium (RPE) function was investigated by direct stimulation with cytokines or indirect stimulation using conditioned medium of polarised human macrophages. IC deposition in the mouse retina led to an inflammatory response that depended on the presence of activating mFcγRs, particularly mFcγRI and mFcγRIII, but not on mFcγRIV. Immune complex deposition and increased numbers of immune cells expressing hFcγRIIa and hFcγRIIb were found in the choroid of early AMD donors and microglia in the retina of wet AMD donor eyes. Finally, macrophage activation differentially impacted on RPE cell function, with regards to barrier function and VEGF secretion. The results in this thesis support the hypothesis that immune complex-mediated inflammation could play a role in the pathogenesis of AMD.

No description available.

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.

No description available.

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.

Allergic contact dermatitis (ACD) is a prevalent skin condition caused by chemical haptens, which enter the epidermis, and by modifying self-proteins, render them immunogenic via the activation of hapten-specific T-cells. It is currently not known which specific protein modifications are responsible for sensitization. This work investigates the extreme sensitizer DNCB, which confers a dinitrophenyl (DNP) protein modification. Immortalised keratinocytes (HaCaT), incubated with DNCB were compared to ex vivo human skin using immunofluorescence and western blot detection of DNP-proteins, showing widespread protein modification and similarities between tissue and cells when using a clinical dose. Proliferation assays using lymphocytes from DNCB-sensitive donors showed responses to DNP proteins isolated from DNCB-treated HaCaT cells and primary keratinocytes. While western blot analysis of pH gradient separated fractions identified a number of DNP-proteins in the DNP-HaCaT cell lysates, these were not immunogenic in lymphocyte assays. The model protein human serum albumin (HSA) was used to investigate the modification kinetics of DNCB by identifying which amino acid residues were changed more readily using a range of DNCB concentrations and incubation times. Cysteine residues, including those in disulphide bonds and particularly cysteine 34 are more readily modified than lysine in HSA, suggesting that DNCB is able to alter the structure of proteins. A novel process of hapten-reversal by a process termed ‘thiolysis’ was found to remove DNP groups from the cysteine residues of synthetic peptides derived from the sequence of HSA containing cysteine 34 using the reducing agent dithiothreitol. Identical peptides with C34>K34 showed no such hapten-reversal. This corresponds to the unexpected immunogenicity of the cysteinyl peptide and also the DNP modified tripeptide glutathione. Anti-DNP western blots show that the DNP group is transferred to other proteins during incubation with human monocytes in culture. This suggests a cellular process of removing DNP groups and GILT, a thiol reductase present in the endosomes is presented as a candidate for this process. This work demonstrates that DNCB can generate a wide variety of DNP protein adducts and that cysteinyl moieties are able to stimulate lymphocyte proliferation by way of hapten transfer. This highlights a potentially novel process involved in the mechanism of contact allergy.

No description available.