The human chemokine family consists of around 50 peptides that control the migratory patterns and positioning of all leukocytes. One such member of this family is CXCL14. Very highly expressed in many healthy tissues including skin, gut and kidney, loss of CXCL14 expression in chronic inflammatory conditions and certain forms of cancer has led to a proposed role for CXCL14 in immune surveillance at these sites. The function and target cells of CXCL14 are poorly defined however, largely because the identity of its receptor remains unknown. Here, I have combined the evaluation of chemotactic responses toward CXCL14 with detection of putative CXCL14 receptor(s) on the surface of cells using a synthetic, fluorochrome-conjugated CXCL14, to definitively identify CXCL14 target cells in human. Monocytes were identified as the major target cells in peripheral blood, 28.4 ± 6.1% Monocytes migrating toward 1 µM CXCL14 in ex vivo transwell chemotaxis assays compared to 3.01 ± 0.65% toward buffer alone (p=0.0031). Responses to CXCL14 also identified tissue phagocytes extracted from healthy human skin, including an apparently novel population of skin-resident CD14+ cells characterised by lack of CD45 expression. Screening of CXCL14-responsive cells by RNA sequencing for expression of G protein-coupled receptors revealed five major candidates for the CXCL14 receptor, all of which are orphan receptors; GPR35, GPR68, GPR84, GPR141 and GPR183. At present, I am in the process of testing these candidates in functional assays. Finally, I report on a novel ability of CXCL14 to potently synergise with other chemokines, particularly CXCL12. This ‘synergy’ with CXCL12 likely occurs via a direct interaction between CXCL14 and the receptor for CXCL12, CXCR4, which is broadly expressed on immune cells. This work identifies mononuclear phagocytes in blood and tissue as the primary targets for CXCL14, providing new and exciting insights into the role played by CXCL14 in immune surveillance of peripheral tissues.

Platelet-derived eicosanoids play important signalling roles in haemostasis and innate immunity and have major medical implications in cardiovascular disease, inflammation and cancer. Therefore, identification and characterisation of novel platelet lipids is an important goal. Prior to my PhD, our group identified a novel cyclooxygenase-1 (COX-1) product generated by platelets upon agonist activation in an aspirin–sensitive manner. Preliminary structural data suggested this lipid to be 8-hydroxy-9,11-dioxolane eicosatrienoic acid (DXA3) (Aldrovandi, unpublished). However, this required further investigation and its biology was unknown. In this study, I undertook a detailed structural characterisation of DXA3. Using GC/MS, high resolution LC/MSn and UV enabled structural characterisation of the lipid. Through pharmacological studies on isolated platelets and using mutant COX-2 enzymes, I revealed a distinct enzymatic mechanism of COX where the dioxolane forms within the COX catalytic site, then a radical escapes from the enzyme to form DXA3 possibly via a peroxidase. I developed a quantitative assay and showed that DXA3 is generated in ng amounts by thrombin-activated platelets (human and murine) with 77% of DXA3 esterified to phosphatidylethanolamine (PE). In addition to platelets, I detected the lipid in human serum derived from whole blood following clot formation in vitro and the macrophage cell line RAW246.7 in response to calcium ionophore A23187 stimulation. Last, DXA3 stimulated expression of the integrin Mac-1 on human neutrophils. In conclusion, DXA3 represents a novel eicosanoid, generated by activated platelets that activates neutrophils and may play a role in early immune responses.

Introduction: There is emphasis on increasing patients’ Physical Activity (PA) to reduce disability and promote independent living. Therefore a new computerised system based on real time location technology called the Rehabilitation Mobility Measurement System (RMMS) was developed to overcome limitations of the current activity monitoring methods and measure PA continuously and unobtrusively. The study objectives were to evaluate the psychometric properties of RMMS and to explore early stage functional recovery after stroke in a rehabilitation unit and at home. Methods: Each participant wore a radio-frequency identification tag with an in-built motion sensor on their unaffected wrist. Walking-aids and transport equipment were also fitted with tags. All areas accessed by patients were fitted with infra-red room locators. The tags transmitted movement and location signals to a computer having customised software programs for data processing. Descriptive statistics and graphs were used for analysis. Results: The RMMS was very reliable (all ICC > 0.90) and demonstrated high level of agreement on validation with observational methods. Longitudinal PA was measured successfully in the rehabilitation unit for 52 patients over 64±53 days. Outside of therapy sessions, patients spent 85% of the waking day in their own rooms undertaking limited high level activities (15%).The average mobility (walking or moving around) was 15 minutes per day only and was strongly correlated with Barthel Index and modified Rivermead Index scores on discharge (spearman’s rho=.-70, p=0.00) accounting for ≥ 43% of variation in these scores. Conclusion: RMMS was a reliable and valid tool for measuring mobility; a key factor influencing early stroke recovery. The small amount of time spent active strongly suggests that better organisation of time outside therapy sessions is warranted to maximise daily PA of in-patients. RMMS could be used for motivational feedback for patients and clinicians to ultimately enhance functional activity during rehabilitation in a stroke unit.

This thesis is a combination of three interlinking projects exploring the association of gestational age at birth with abnormal vascular function measurements, increased respiratory symptoms and reduced exercise capacity in childhood. The main aim of my thesis was to identify any evidence of early disease processes (respiratory or vascular) in children born preterm. My first objective was to investigate the association between preterm birth and markers of vascular disease in childhood using data from Avon Longitudinal Study of Parents and Children (ALSPAC), and to review the literature reporting on arterial stiffness and/or endothelial dysfunction in preterm-born children. In my second project I conducted a systematic review of the literature with the objective to answer the question: Do survivors of preterm birth have reduced cardiorespiratory exercise capacity? My final project was to set up a cross-sectional study of children in Wales to test if lower gestational age at birth is associated with increased respiratory symptoms, admission rates to hospital and inhaler use for respiratory disease; independent of atopy. My first research project, using data from ALSPAC, noted an association between gestational age and SBP but not with measures of vascular function in childhood suggesting that alternative mechanisms may be responsible for increased SBP in preterm-born children. These results were supported by the findings of the seventeen articles identified by my systematic review. In my second project, a systematic review of the literature, I noted that despite marked deficits in lung spirometry, preterm-born children have only marginally decreased VO2max, which is unlikely to be of great clinical significance. Finally in my cross-sectional survey, I showed that increasing prematurity, and children who were born at early term, are associated with increased respiratory symptoms and utilization of healthcare services throughout childhood, which is independent of a family history of atopy or mode of delivery.

A dry cough is a common symptom described in patients with IPF and impairs quality of life. The exact mechanisms causing cough in IPF remain unclear, however there is evidence that altered cough neurophysiology and sensitisation plays a roleY IPF patients have an enhanced cough reflex sensitivity to the inhalation of capsaicin. It was hypothesised that IPF patients have increased airway expression of the capsaicin receptor TRPVF1 and a coFexpressed receptor TRPAF1. Bronchial biopsies were obtained in 16 IPF patients, 11 chronic cough patients and 8 controls. Quantitative PCR was used to detect TRPVF1 and TRPAF1 gene expression, with immunohistochemistry demonstrating protein expression. Mean TRPVF1 and TRPAF1 gene expression was higher in IPF patients compared with controls, but the difference did not reach statistical significance. Immunostaining supported these findings. Gastroesophageal reflux is common in IPF patients and has also been implicated. An inFvitro study using cultured pulmonary epithelial cells was conducted to assess the expression of these receptors in a cell model of gastric reflux. TRPVF1 and TRPAF1 gene expression was demonstrated in pulmonary epithelial cells of bronchial and alveolar origin. No significant difference in receptor expression level was seen in either cell line when exposed to the major constituents of gastric refluxate. This study suggests that a structural upFregulation of central airway TRP receptors is not the key mechanism for cough in IPF patients. Similarly, it does not support the role of the individual constituents of gastric refluxate resulting in cough hypersensitivity through a physical upFregulation of receptors in pulmonary epithelial cells. Overall this thesis outlines the complexity of the cough reflex. It is probable that cough in IPF results from the cumulative manifestation of various physiological changes and mechanisms.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in equine veterinary practice. These drugs exert their effect by inhibiting cyclooxygenase (COX) enzymes, which control prostaglandin production, a major regulator of tissue perfusion. Two isoforms of COX enzymes exist: COX-1 is physiologically present in tissues, while COX-2 is up-regulated during inflammation and has been indicated as responsible for the negative effects of an inflammatory response. Evidence suggests that NSAIDs that inhibit only COX-2, preserving the physiological function of COX-1 might have a safer profile. Studies that evaluate the effect of NSAIDs on COX enzymes are all performed under experimental conditions and none uses actual clinical patients. The biochemical investigations in this work focus on describing the effect on COX enzymes activity of flunixin meglumine and phenylbutazone, two non-selective COX inhibitors and firocoxib, a COX-2 selective inhibitor, in clinical patients undergoing elective surgery. A separate epidemiological investigation was aimed at describing the impact that the findings of biochemical data have on a large population of equids. Electronic medical records (EMRs) from 454,153 equids were obtained from practices in the United Kingdom, United States of America and Canada. Information on prevalence and indications for NSAIDs use was extracted from the EMRs via a text mining technique, improved from the literature and described and validated within this Thesis. Further the prevalence of a clinical sign compatible with NSAID toxicity, such as diarrhoea, is reported along with analysis evaluating NSAID administration in light of concurrent administration of other drugs and comorbidities. This work confirms findings from experimental settings that NSAIDs firocoxib is COX-2 selective and that flunixin meglumine and phenylbutazone are non-selective COX inhibitors and therefore their administration carries a greater risk of toxicity. However the impact of this finding needs to be interpreted with caution as epidemiological data suggest that the prevalence of toxicity is in fact small and the use of these drugs at the labelled dose is quite safe.

This thesis is concerned with the measurement of locomotor disability (LMD) in epidemiological studies. The central hypothesis was that LMD is a continuous phenomenon and research into this important health indicator, with specific reference to its relationship to pain in community-dwelling adults aged 50+ years, could be improved by interval-level measurement, rather than binary definitions. A systematic search and narrative review of the literature revealed a range of concepts and content of previous self-complete LMD instruments, and an absence of interval-level measures. A brief, self-complete scale of physical functioning, the PF-10, commonly used in epidemiological studies, and suggested as a measure of LMD, was taken as the starting point for empirical work in this thesis. A subset of five items mapped onto the LMD construct and possessed acceptable psychometric properties. Analysis of cross-sectional data from 18,497 adults using ordinal regression models and individual item responses illustrated one, albeit relatively inefficient, approach to moving beyond binary outcomes for investigating the association between pain and LMD. An interval-level measure of LMD was derived using the Rasch model and combining the five items into two super-items (walking, stair-climbing). The scoring mechanism was externally verified in local, national and international datasets, and the psychometric properties confirmed. Data from 680 initially pain-free adults were used to demonstrate the potential of the new measure for longitudinal analysis. This suggested a right-shift (worsening) in the distribution of LMD at three and six years. Pain onset resulted in a more rapid increase in LMD, and recovery from pain led to only a partial return to pre-pain levels. Locomotor disability exists on a continuum and its measurement should reflect this. An interval-level measure was derived from a set of commonly used items. This measure offers several advantages (brevity, application to retrospectively gathered data) but also has limitations (ceiling/floor effects).

Malaria, the leading cause of death due to vector-borne disease. is responsible for around 250 million cases and 1 million deaths annually, mostly in sub-Saharan Africa where Anopheles gambiae is the major vector. Novel approaches to malaria control include theoretical use of transgenic mosquitoes to suppress or replace wild type populations. Generation of mosquitoes carrying either a conditional lethal gene (RIDL), or one encoding a molecule able to kill malaria parasites, is a key component of this strategy. Detailed genetic and physiological knowledge of transgenic mosquitoes is important for epidemiological models to accurately determine the outcome of putative field releases. This includes testing the potential for post-integration remobilisation of transgenes in the host genome and testing the reproductive fitness of transgenic mosquitoes in comparison to wild type. Complex plasmids, designed to create both RIDL strains and strains within which piggyBac remobilisation could be tested, were injected into the Keele strain of An. gambiae. Ultimately, although microinjection techniques were optimised and efficiencies improved over time, generation of such transgenic strains was unsuccessful. A previously established transgenic strain (EVida3), expressing the Vida3 peptide which is active against ookinete stages of Plasmodium, was subsequently investigated with respect to multiple feeding and relative fitness. Multiple feeding experiments indicate that complex interactions take place between Vida3 and the natural immune system when mosquitoes are infected with the murine malaria P. yoelii nigeriensis. Vida3 production appears to impact on parasite intensity and mosquito fecundity in both positive and negative ways dependent on factors such as gonotrophic cycle and parasite-stage. Analysis of three life-table fitness parameters, comparing EVida3 hemizygotes to the KIL wild type laboratory strain, indicate no fitness cost associated with transgene presence or expression. Analysis of a further two parameters indicates a possible fitness cost associated with inbreeding rather than transgenesis alone.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by chronic synovial inflammation, ultimately leading to joint destruction and permanent disability. Disease expression may be affected by genetic variations and environmental factors such as cigarette smoking. The main objective of this study was to identify some of the main determinants of poor outcome in RA, both in terms of disease severity and comorbidity. Analysis of possible interaction of genetic factors with smoking was carried out. Candidate genes included GSTM1, GSTT1, VEGFA, eNOS, MMP1, MMP2, MMP3, TGFB1 and PTPN22. Smoking was associated with seropositive RA, in particular with RF+ RA. It was associated with the development of erosive disease and with more severe functional outcome in seronegative patients. Promoter polymorphism VEGFA-2578(A/C) (rs699947) was associated with serum VEGF-A level, which may reflect a genotype-specific response to inflammation. This polymorphism was associated with disease activity, which only occurred in nonsmokers. The same polymorphism was also associated with the occurrence of IHD and MI, which may be due to an interaction with smoking. Both MMP1 (rs1799750) and MMP3 (rs679629, rs3025058) polymorphisms were independently associated with serum MMP-1 level, whereas serum MMP-3 was mainly associated with MMP3 polymorphisms. MMP3 SNPs were associated with disease activity, independent of systemic inflammation and serum MMP-3. A haplotype across the MMP1-3 loci was associated with the development of erosive disease in earlier RA. Evidence of interaction with smoking was also found regarding the above association. No association of polymorphisms in TGFB1 with serum TGF-β1 level was found. A missense polymorphism TGFB1+868(C/T) (rs1800470) was associated with the occurrence of IHD and MI, which may be explained by an interaction with smoking. The current thesis highlighted the complexity of factors associated with severity and comorbidity in RA, and showed the importance of smoking in exacerbating various aspects of disease outcome.

Excess fluid and waste products of metabolism, as well as protein, are removed from the peritoneal cavity in Peritoneal Dialysis (PD). Increased peritoneal protein clearance (Pcl) is associated with a greater risk of mortality. It is not clear whether this association reflects systemic endothelial injury or local peritoneal capillary damage and inflammation, or both. To investigate this problem a series of analyses were undertaken in different incident, prevalent and longitudinal patient cohorts. Transcapillary escape rate of albumin (TERalb) was measured to determine systemic capillary permeability. Luminex assays combined with principle component analysis were applied to measure endothelial biomarker patterns. It was demonstrated that: (1) Pcl is a function of both local peritoneal inflammation, membrane area (PSTR) and comorbidity (especially cardiovascular) but only its association with the latter predicted survival. (2) There is a progressive uncoupling of the Pcl, (indicative of large pore pathway) and PSTR (effectively the small pore area) with time on PD. (3) Isolated small pore ultrafiltration (due to icodextrin) decreases with prolonged time on PD and is also uncoupled from the increase in peritoneal membrane area. (4) The systemic endothelial barrier function is decreased in PD patients, especially diabetics, but not associated with hypoalbuminaemia which is linked to systemic inflammation. (5) Hydration status is related to plasma albumin concentration but not endothelial dysfunction as measured by soluble biomarkers. Pcl is a function of both local peritoneal factors, e.g. inflammation and progressive fibrosis, and systemic patient characteristics, e.g. age and comorbidity. The influence of comorbidity is complex depending on type, associated patterns of endothelial injury and causes of associated hypoalbuminaemia. The importance of plasma colloidal pressure in determining fluid status was emphasized. Strategies to improve fluid distribution should focus on reducing peritoneal protein loss and increasing albumin synthesis rather than blocking systemic vascular leak.