Polysaccharide conjugate vaccines are available to prevent disease caused by Neisseria meningitidis serogroups A, C, W and Y. Meningococcal vaccine efficacy is assessed in clinical trials using serum bactericidal assays (SBAs). Baby rabbit serum is usually used as the source of complement for SBAs as it lacks endogenous bactericidal activity compared to human serum. Previous studies have shown that SBA activities determined with rabbit (rSBA) and human (hSBA) complement correlate poorly, possibly due to different interactions between antibody subclasses and the complement source, and species-specific interaction of Neisseria meningitidis with complement regulators. The aim of this project was to investigate the mechanisms responsible for differential bactericidal activities of human and rabbit complement against Neisseria meningitidis. The serum concentration of polysaccharide-specific antibody subclasses was measured following vaccination with quadrivalent meningococcal polysaccharide vaccines; data showed that the concentration of polysaccharide-specific IgG1 antibody correlated most significantly with hSBA titres whereas the concentration of polysaccharide-specific IgM antibody correlated most significantly with rSBA titres. The interaction of human IgM and IgG subclasses with human and rabbit complement was compared at the level of C1q and C3 using both binding and functional assays. These data define important differences in the ability of human antibody subclasses to fix human and rabbit complement. Specifically, polysaccharide-specific IgM contributes significantly more to bactericidal titres in rSBAs compared to hSBAs. As a consequence, rSBAs produce misleadingly high titres in individuals with large IgM responses to vaccination. Using a series of pathway-specific inhibitors, it was shown that the alternative pathway contributes significantly more to the bactericidal activity of rabbit complement towards Neisseria meningitidis than human complement. This project provides significant insight into the difficulties and challenges associated with the interpretation of rSBA data, enhances the understanding of antibody responses to meningococcal vaccines and will support improvements in the development and testing of meningococcal vaccines.

Clopidogrel, prasugrel, and ticagrelor are antiplatelet agents used for the treatment of acute coronary syndromes. Clopidogrel is known to improve endothelial function in patients with coronary disease but little is known about either the more potent thienopyridine prasugrel, or the irreversible P2Y12 inhibitor ticagrelor. The ability of clopidogrel to undergo S-nitrosation is recognised, and the thienopyridines’ ability to form S-nitrosothiols (RSNO) has been confirmed in vitro, a finding of significant interest given the potent anti-aggregatory and vasomodulatory properties exhibited by S-nitrosothiols. This study sought to investigate firstly, the effect of co-administration of oral nitrates and proton pump inhibitors on NO metabolites in patients treated with clopidogrel. Secondly, the effect of acute and chronic prasugrel treatment on NO metabolite formation was investigated, with particular emphasis on SNO bio-synthesis in-vivo. This lead to further interest in ticagrelor which, unlike the thienopyridines, lacks a free thiol group, to examine the effect of changing pH on its ability to dissolve, react and inhibit platelets, and ultimately establish whether ticagrelor could form RSNO. Ozone-based chemiluminescence techniques were employed to measure the principal NO metabolites in blood samples, and platelet aggregation was measured using multiple electrode aggregometry. The ability of clopidogrel and prasugrel to form RSNO is demonstrated both in vitro and in vivo. An acute rise in plasma RSNO levels occurs following a loading dose of prasugrel in patients with coronary disease. Ticagrelor’s platelet inhibitory response to ADP was found to decrease after lowering of the pH in vitro. However in the presence of nitrite and decreasing pH, it readily formed ticagrelor-induced RSNO which resulted in augmented platelet inhibition compared to ticagrelor alone. These are exciting and novel findings with the potential to shape both our understanding of RSNO, and the pleiotropic effects of these commonly prescribed anti-platelet drugs.

Enhanced bone resorption is a common pathology in destructive bone diseases. Many cytokines (e.g. IL-6 and TNF-α) and chemokines (e.g. CCL2, CCL3 and CCL5) elevated in patients exert pathological roles in leukocyte migration and inflammation, but their effect on direct bone destruction remained elusive. Published research into osteoclastogenesis was carried out in co-cultures, from which osteoclast differentiation, resorption and mediator secretion data was obtained. The main objective of this thesis was to establish a working methodology for the in vitro differentiation of CD14+ve mononuclear cells into osteoclasts and to decipher the direct effects of IL-6 and CCL3 on osteoclastogenesis and bone resorption. IL-6 trans-signalling exerted an effect in both basal and pathological osteoclastogenesis, whereas its inhibition via sgp130-Fc significantly reduced osteoclast formation and bone resorption in vitro. Although not examined in vivo, the translational use of sgp130-Fc as a therapeutic for destructive bone pathology was postulated from data showing a reduction in osteoclast differentiation and resorption after stimulation with HYPER-IL-6. Secondary to IL-6 trans-signalling, it was hypothesised that the neutralisation of CCL3 in vitro and in vivo would significantly reduce osteoclast differentiation and resorption. Osteoclast number significantly reduced in the presence of anti-CCL3, but TRAP+ve cell count was unaltered, suggesting an early role of CCL3 in osteoclast multi-nucleation/fusion. Additionally, in vivo data showed a protective effect of anti-CCL3 with significantly reduced bone erosive scores and osteoclast counts thereby presenting CCL3 as a novel biomarker of disease activity in destructive bone disease. In contrast, TNF-α, CCL2, and CCL5 were shown to have no role in direct osteoclast differentiation in our monocultures. In conclusion, for the first time this work documents novel and important roles of IL-6 trans-signalling and CCL3 in increased osteoclast differentiation and bone resorption. Our data highlights the importance of IL-6 trans-signalling and provides evidence for the use of CCL3 as a predictive biomarker in destructive bone diseases.

Background RA patients have an increased prevalence of CVD independently of traditional risk factors. Studies using a mouse model of inflammatory arthritis – mCIA have shown decreased vascular constriction response of the thoracic aorta to 5HT, associated with increased MMP-9 production. The source of the latter, inflammatory content and impact on the structural proteins of the vessel wall remains elusive. Methods Myography was used to determine vascular constriction response in mCIA animals. Immunohistochemistry determined presence of F4/80+ macrophages, Ly6G+ neutrophils, DR3 and MMP-9. DR3 was assessed in the healthy and arthritic constriction response using DR3-/- and DR3WTs. Vascular calcification was determined in short and long term mCIA using RT-qPCR. Protein levels were quantified using immunohistochemistry. Collagen and elastin were determined using Van Geisson and Ver Hoeffs staining. The role of the AIM2 inflammasome in mCIA associated vascular dysfunction was also determined using CRID3 therapy. Results Increased macrophages with complimentary DR3 staining were observed in the aorta and PVAT.DR3-/- had normal constriction response despite increased cells and MMP-9 in the PVAT. DR3 ablation decreased arthritis onset and severity, however, worsened the vascular function. DR3 PVAT was protective to the constriction response. Calcification mediators remained constant following the onset of mCIA. Long term mCIA showed exciting trends of increase to calcification mediators. Collagen and elastin both become deregulated during mCIA and showed a fibrosis like phenotype. Blocking the AIM2 inflammasome had no impact on mCIA onset but partially restored vascular constriction response. Discussion Systemic inflammation is key in explaining the vascular dysfunction associated with the mCIA model. mCIA allows us to determine very early changes in the vasculature associated with systemic inflammation as opposed to long term changes. Macrophages specifically are early inflammatory cells implicated in the aorta and are associated with both DR3 and AIM2, suggesting them as key players contributing to vascular dysfunction in this model. Successful AIM2 blocking therapy has potential to be used in human RA patients to reduce CV co-morbidity.

Poor adherence to medication wastes resources and can lead to reduced exposure to and effectiveness of pharmacological treatments. Poor adherence to medication in clinical research can dilute treatment effects, obscuring the true benefits that medication can provide. The study of medication adherence comprises significant methodological challenges. The aim of my thesis was to investigate several methodological challenges encountered when studying medication adherence in clinical research using data from five clinical studies. Several methods for measuring adherence were compared using both correlation and agreement approaches. I proposed extensions to data visualisation techniques for comparing agreement. As an alternative to reporting summary measures, I explored the use of advanced modelling techniques to model adherence data collected via electronic monitors. I also moved beyond comparisons of measures and investigated approaches for predicting disagreement and calibration techniques. I investigated various methods for modelling the determinants of adherence, considering determinants according to type of measure used, type of condition being studied, different study designs, and different conceptualisations of adherence. I explored, quantitatively, the extent to which the treating clinician influenced whether a patient adhered to their treatment. I also established the feasibility of calculating randomisation-based efficacy estimators in randomised controlled trials with non-adherence, scrutinising the implementation of these approaches during placebo-controlled trials and non-inferiority trials involving two active treatments. My findings emphasise the need for considering the impact of medication adherence when designing a study, rather than leaving it as an afterthought, as it would appear to be much of the time. Such considerations include selecting an appropriate mode (or modes) of medication adherence ascertainment, agreeing adherence definitions of interest, measuring variables that are likely to be associated with adherence, and, particularly for trials, determining whether it is feasible to adjust findings for non-adherence while maintaining a comparison of groups as randomised.

Wound healing and the management of chronic, non-healing wounds represent a significant burden to the NHS and results in substantial patient morbidity. New methods to further understanding of wound chronicity and potential therapies are needed. This PhD study aims to explore the importance of the SOCS family in the wound healing process, exploring their potential to act as prognostic factors in clinical cohorts and exploring the potential of SOCS-3 and -4 to influence key cellular traits linked to the healing process. Detection of SOCS family members within a clinical healing/non-healing cohort highlighted significant elevations of gene expression of SOCS-3 and -4 in non-healing chronic wounds compared to healing chronic wounds, though this trend was no as obvious in a smaller cohort of IHC stained samples. However, the IHC stained samples potentially indicated that SOCS-3 protein relocalisation, from wound edge to distal wound area, was evident in the non-healing chronic wound. Subsequently, SOCS-3 expression and SOCS-4 knockdown lines were generated in human (HaCaT) keratinocytes and (HECV) endothelial cells to explore the functional significance of these molecules at a cellular level. In summary, SOCS-4 knockdown decreased the migration of both HaCaT and HECV cells on plastic cultureware, whereas downregulation of SOCS-4 only reduced the adhesion and tubule formation of HECV cells on matrigel matrix. SOCS-3 expression attenuated the proliferation of HaCaT cells but had no effect on HECV cells. In addition, SOCS-3 upregulation solely improved the adhesion and tubule formation of HECV cells on matrigel matrix. Moreover, the regulatory role of SOCS-3 and SOCS-4 in the migration and adhesion of HaCaT and HECV are likely to be substratum matrix dependent. Finally, to explore potential mechanisms of action for SOCS-4 role in HaCaT cell line, a protein microarray was utilised to highlight a number of potential key pathways. Based on the results from protein microarray and wound healing assay as well as evidences from literature review, four potential mechanisms (FAK/Src/p130cas, HBEGF/EGFR, VEGF/VEGFR-2, IGF-1/IGF-1R/PI3K) of SOCS-4 relulating keratinocyte migration was highlighted for further validation, and the expression profile of FAK Y397 was varified by western blotting. Hence, SOCS-3 and SOCS-4 may regulate keratinocyte and endothelial cell behaviour, and potentially have effect on the wound healing process.

Introduction: Chronic wounds do not progress through the normal wound healing process and represent a significant burden to healthcare systems and to the hundreds of thousands of patients in the UK who suffer with them. There are significant challenges in both understanding the underlying biology and developing novel therapies to encourage healing of chronic wounds. nWASP is a regulator of the actin cytoskeleton through interactions with the Arp2/3 complex and is involved in a variety of roles particularly at the cell membrane in protrusion and vesicle formation. This study investigates the expression pattern of nWASP in chronic wound tissues and explores, through both in vitro and in vivo models, the cellular and molecular impact of nWASP activity on the function of cells involved in wound healing and also in cancer cells. Methods: Clinical cohorts from patients with chronic wounds and lung cancer were tested for the expression of nWASP expression through qPCR analysis. Cell models, based on keratinocytes (HaCaT) and vascular endothelial (HECV) cells were employed to evaluate the influence of nWASP on cellular functions that are key to the healing process genetic knockdown and/or using nWASP-specific inhibitors. Resulting cellular signalling changes was explored by way of protein kinase arrays and Western blotting and in vivo models were used to assess the therapeutic values of topical application of nWASP inhibitors in wound healing. A similar approach was employed using lung cancer cell models. Results: nWASP was found to be significantly elevated at the transcript level in human non-healing chronic wound tissues compared with healing tissues. nWASP inhibitors, namely wiskostatin and 187-1, and knockdown, altered the spreading and attachment behaviour of HaCaT cells. Several protein signalling pathways affected by nWASP inhibition were identified, in particular TrkB signalling and downstream PLCγ1 phosphorylation were shown to be impaired by nWASP inhibition in HaCaT cells. Healing of hole-punch wounds in the ears of db/db mice, a diabetic model which exhibit impaired wound healing, was improved by topical and systemic nWASP inhibitor treatment. In the context of lung cancer, nWASP expression was significantly correlated with the survival of the patients. Invasive behaviour was found to be impaired in the more invasive SK MES-1 cell line with motility and migration being affected in A-549 cells following nWASP inhibitor treatment. Decreased paxillin activity in membrane focal adhesions was shown following nWASP inhibition and knockdown. Conclusions: This study suggests that nWASP activity may be related to the nonhealing behaviour of chronic wounds and that its activity can affect function of keratinocytes and endothelial cells. Together with the findings in the in vivo models, it strongly points nWASP as a therapeutic target in non-healing wounds. nWASP is also a potential biomarker and therapeutic target in human lung cancer, in which it influences the aggressive behaviour of lung cancer cells. The study has identified that TrkB, PLCγ1 and paxillin signalling can be controlled by nWASP activity.

No description available.

In search of genes differentially expressed between M1 (pro‐Th1 or pro‐inflammatory) and M2 (pro‐Th2 or pro‐tolerogenic) macrophages, BIC (microRNA 155 hosting gene) was found up regulated under inflammatory conditions. MicroRNAs are non coding RNAs of ~22nt in length that inhibit gene expression upon pairing to the 3’UTR (UnTranslated Region) of target mRNAs. In silico analysis predicted two pro‐Th2 targets for miR‐155: PU.1 and IL13R1. PU.1 is a transcription factor essential in myelopoiesis and dendritic cells (DCs) that favours a Th2 profiling; moreover, PU.1 had been shown to regulate the transcription of DC‐SIGN (Dendritic Cell‐ Specific ICAM‐3 Grabbing Non‐integrin 1) which is a pathogen receptor expressed in DCs controlled by Th2 stimuli. IL13R1 is the chain receptor for the Th2 cytokine IL‐13, which promotes M2 differentiation. Pro Th1 stimuli cause maturation of DCs, cells that orchestrate the immune response between Th1 and Th2 profiles; moreover, Th1 stimuli cause classical (M1) macrophages activation versus an alternative (M2) one. My hypothesis was that miR‐155 contributes to the pro‐Th1 profile by down regulating pro‐Th2 factors. MiR‐155 was found up regulated during DC maturation and both PU.1 and IL13R1 were demonstrated as direct targets of miR‐155. Employing a developed monocytic cell line which harbors a miR‐155 transgene under the control of a Tet‐On system (THP1‐155 cells), both PU.1 and IL13R1 were shown to be down regulated following miR‐155 induction in these cells. Moreover, THP1‐155 cells showed that DC‐SIGN transcription is regulated by miR‐155 levels through PU.1 targeting, and that IL‐13 signalling cascade through STAT6 transcription factor was down regulated when miR‐155 was over expressed. Using Anti miR‐155 transfections in DCs, miR‐155 was shown to modulate not only DC‐SIGN expression, but also DC pathogen binding ability. Using the same technique in macrophages, miR‐155 was shown to modulate IL13R1 and STAT6 activation, and to regulate the expression of IL‐13/STAT6 dependent genes. Therefore, miR‐155 contributes to the pro‐Th1 profile by down regulating pro‐Th2 factors, acting as a pro‐Th1/anti‐Th2 modulator in myeloid cells under inflammatory conditions

Members of the tumour necrosis factor superfamily (TNFSF) are important regulators of inflammation and immunity. TL1A (TNFSF15), ligand for death receptor 3 (DR3), is the most recently discovered member of this superfamily and full understanding of its structure and the role in T cell-mediated immune responses is currently incomplete. DR3 expression is strongly up-regulated on activated T cells, although it also is present on resting CD4+ T cells, while the expression of TL1A is rapidly and transiently upregulated on activated cells of the immune system such as dendritic cells, monocytes and T cells. The research published to date shows that TL1A/DR3 interaction is involved in the pathogenesis of several autoimmune diseases and enhances activation of CD4+ T cells, however very little is known about its role in co-stimulation of CD8+ T cells. Several studies showed that TL1A also acts as a polarizer of the immune response by inducing secretion of IFN-γ, IL-4, IL-10 and/or IL-17A from activated CD4+ T cells, although the results vary depending on the conditions of a given experiment. The research presented in this thesis identifies Toll-like receptors 3 and 4 as the inducers of TL1A expression on dendritic cells. Furthermore, different binding patterns of anti- TL1A monoclonal antibody (raised against the homotrimeric form of TL1A) and DR3.Fc construct to cells transfected with TL1A cDNA and cells naturally expressing TL1A suggest that TL1A may exist as a homo- and heterotrimeric protein. Ecotopic expression of TL1A on J558L tumour cells promotes their elimination in a CD8+ T celldependent manner and renders mice immune to a subsequent challenge with tumour cells. Moreover, TL1A promotes the proliferation and accumulation of antigen-specific CD8+ T cells both in vitro and in vivo as well as their activation and differentiation into cytotoxic T cells in vivo. It also enhances the secondary expansion of endogenous antigen-specific memory CD8+ T cells. The studies presented here also show that TL1A/DR3 interaction enhances the proliferation and activation of CD4+ T cells. CD11c-TL1A transgenic and CD2-TL1A transgenic mice that constitutively express TL1A on dendritic cells and T cells, respectively, show elevated levels of IL-13 and IL- 17A in the secondary lymphoid organs suggesting that in this setting TL1A skews the immune response toward Th2 and Th17 type. Furthermore, CD11c-TL1A transgenic mice develop a striking goblet cell hyperplasia in the ileum. TL1A also enhances regulatory T cell accumulation in vivo. The findings presented in this thesis show that TL1A may have the potential for enhancing vaccines that aim to elicit CD8+ T cell responses and also identify mechanisms by which sustained expression of TL1A could promote pathogenesis in inflammatory bowel disease