Lung cancer is the most commonly diagnosed cancer and the most common cause of cancer related death worldwide. Population-based lung cancer screening programmes have been initiated in the USA and could soon be implemented in other countries. The overarching purpose of this thesis was to explore the emotional impact of lung cancer screening. The research was conducted as part of a clinical trial that was investigating the effectiveness of a blood autoantibody test, EarlyCDT®-Lung, in identifying individuals at the risk of lung cancer. A systematic review was conducted that aimed to identify factors associated with the emotional impact of screening for lung cancer. Participants with indeterminate test results, current smokers and females were more likely to experience negative non-specific and specific emotional outcomes. In addition to highlighting several key factors associated with higher levels of emotional distress following screening, factors that warranted further research were also identified. Such factors included age, education level, marital status, ethnic origin, and perceived risk of developing lung cancer. Finally, important methodological and theoretical limitations in the literature were identified. One key methodological limitation was that no studies measured positive emotional outcomes. A longitudinal study was conducted exploring the impact of lung cancer screening on positive affect, negative affect, lung cancer worry and distress specific to screening for lung cancer. Participants from each of the EarlyCDT®-positive, EarlyCDT®-negative, and control groups completed questionnaires containing emotional outcome measures at pre-randomisation and then at one, three, six and 12 months post randomisation. Scores for each outcome measure were described by groups over time and multilevel regression modelling was used to compare scores over time within and between groups. Results were reassuring as screening was found to have no clinically important impact on positive affect, negative affect, frequency of lung cancer worry or impact of lung cancer worry on mood and ability to perform daily activities. Although screening specific distress in the EarlyCDT®-positive group was significantly higher than that of the EarlyCDT®-negative group, it did reduce over time. Statistically significant and clinically important increases in the proportion of participants reporting anxiety about the results of future tests/treatments were identified. As a result of this finding, a further study was carried out to identify factors that could influence an individual’s level of anxiety about the results of future tests/treatment. Participants more at risk of reporting anxiety about the results of future tests/treatment were younger participants, non-white participants, current smokers and participants who did not own or have a mortgage on their home. Psychological variables associated with increased anxiety were: higher general anxiety scores, higher depression scores, higher negative affect scores, participants who reported that they were upset when they thought about their risk of lung cancer, participants who were worried about getting lung cancer, and those who reported the highest impact of lung cancer worry on mood and ability to perform daily activities. The final chapter of this thesis presents the results of a randomised controlled trial embedded within the emotional outcomes study (described above), which evaluated the effect of timing of monetary incentives (£5 voucher sent with questionnaire vs. £5 voucher sent on receipt of questionnaire) on the following outcomes: study participation rates, questionnaire response rates over time, the number of reminders sent and the completeness of returned questionnaires over time. Previous research had found that monetary incentives were useful in increasing response rates in clinical trials. Results from this trial extended the evidence base by showing that the timing of monetary incentives makes no difference to the above outcomes. In each chapter the findings of this thesis are discussed in terms of their contribution to knowledge. Recommendations for future research and clinical practice are also made within each chapter.

Introduction Airway remodelling describes the histopathological changes in tissue architecture observed in obstructive lung diseases such as asthma and may have a negative impact on lung function. These changes do not appear to be treated by current asthma treatments. Changes observed during airway remodelling include increased thickness of airway smooth muscle (ASM) layer and enhanced extracellular matrix (ECM) deposition. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes, which facilitate tissue remodelling via ECM protein degradation. Matrix metalloproteinase-1 (MMP-1) and mast cells are present in the airways of patients with asthma (but not in healthy people). MMPs expression is highly regulated in lungs and is increased in disease states. My project aimed to assess MMP-1, -2 and -9 expression and activity in asthma airways. Furthermore, the underlying mechanism of MMP-1 activation and subsequently its role in airway remodelling and worsening asthma severity was investigated in the context of asthma exacerbation, which is thought to be an exaggerated lower airway inflammatory response to an environmental exposure such as respiratory virus infection. Methods Patients with stable asthma and healthy controls underwent spirometry, methacholine airway (PC20 ) challenge, exhaled nitric oxide (FeNO) test, bronchoscopy/bronchial washings and primary airway smooth muscle (ASM) cell cultures. A second asthma group (mild to moderate severity) and controls had symptom scores, spirometry and bronchoalveolar lavage (BAL) before and after rhinovirus inoculation. ECM was prepared from decellularised primary ASM cultures. MMP-1 protein levels and activity were assessed in bronchial fluid samples by enzyme-linked immunosorbent assay (ELISA), western blotting and fluorescent activity assay. ASM cell growth was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reduction assay and cell counts. Bronchial fluid gelatinase (MMP-2 and -9) expression and activity was assessed by gelatin zymography. Results MMP-1 and MMP-9 expression was enhanced in both stable asthma and during asthma exacerbations, whilst MMP-2 expression was only increased during asthma exacerbations. MMP-1 can be activated by tryptase, which is an inflammatory product of mast cell degranulation. Activated (degranulated) mast cells enhanced proliferation of both control and asthma ASM cells via the production of a pro-proliferative ECM in vitro and the proliferative effect was dependent on MMP-1. In patients with asthma, mast cells numbers within ASM bundles were associated with ASM growth. MMP-1 protein levels were related to bronchial reactivity and MMP-1 activity increased during asthma exacerbations, where its levels were related to exacerbation severity. Conclusion This study suggests that MMP-1 plays an important role in asthma pathophysiology and that ASM/mast cell interactions contribute to asthma severity by transiently increasing MMP-1 activation, ASM growth and airway responsiveness. Moreover, there is increased expression of MMP-2 and -9 during asthma exacerbations compared with stable asthma. As both MMP-2 and -9 act as mediators of inflammation (Okada, S. et al., 1997) (Elkington, P.T.G., 2006) and tissue remodelling (Oshita, Y. et al., 2003), an increase in gelatinolytic activity linked to MMP-2 and MMP-9 is also likely to play a significant role in the pathophysiology of asthma exacerbations.

Genome-wide association studies of human lung function and Chronic Obstructive Pulmonary Disease have identified a highly significant and reproducible signal on 4q24. It remains unclear which of the two candidate genes within this locus may regulate lung function: GSTCD, a gene with unknown function, and/or INTS12, a member of the Integrator Complex which is currently thought to mediate 3’end processing of small nuclear RNAs. An interrogation of bioinformatic datasets showed that in lung tissue, 4q24 polymorphisms associated with lung function correlate with INTS12 but not neighboring GSTCD expression. In contrast to the previous reports in other species, a minor alteration of small nuclear RNA processing was observed following INTS12 depletion. RNA sequencing analysis of knockdown cells instead revealed dysregulation of a core subset of genes relevant to airway biology and a robust downregulation of protein synthesis pathways. Consistent with this, protein translation was decreased in INTS12 knockdown cells. In addition, chromatin immunoprecipitation and sequencing experiments demonstrated INTS12 binding throughout the genome, which was enriched in transcriptionally active regions. Finally, INTS12 regulome was defined which includes genes belonging to the protein synthesis pathways. INTS12 has functions beyond the canonical snRNA processing and evidence is presented showing that it regulates translation by directly controlling the expression of genes belonging to protein synthesis pathways. This thesis provides a detailed analysis of INTS12 activities on a genome-wide scale and contributes to the understanding of biology behind the genetic association for lung function at the 4q24.

Background Evidence from the UK suggests the incidence of idiopathic pulmonary fibrosis is increasing, but there is a lack of data from elsewhere in the World. The cause of the disease remains unknown. New anti-fibrotic therapies may increase the use of surgical lung biopsy for accurate diagnosis, although the risks of this (and other surgery) are not clear. Methods Collated international mortality statistics and a systematic review of the literature were used to assess the incidence and mortality of idiopathic pulmonary fibrosis worldwide. Primary care data from the United Kingdom were used to assess the association between recent major surgery and a new diagnosis of idiopathic pulmonary fibrosis. Secondary care data from the United States and United Kingdom were used to assess the risk of surgical lung biopsy for the diagnosis of interstitial lung disease, and the risk of other major surgery in those with idiopathic pulmonary fibrosis. Results Mortality from idiopathic pulmonary fibrosis in increasing steadily worldwide. Incidence varies worldwide but is in the range of 3-9 per 100,000 in the West. No association was identified between recent major surgery and a new diagnosis of idiopathic pulmonary fibrosis. Surgical lung biopsy for the diagnosis of interstitial lung disease has an in-hospital mortality of under 2% for elective procedures, but this is higher for non-elective surgery, and in those who are older with co-morbidities. In those with idiopathic pulmonary fibrosis undergoing major surgery, in-hospital mortality was higher than the general population. Conclusion Idiopathic pulmonary fibrosis seems to be increasingly common worldwide. Surgery has risks, particularly in unwell older patients, and less invasive diagnostic methods are needed.

Rationale Chronic obstructive pulmonary disease (COPD) is a progressive lung condition with extrapulmonary manifestations- cardiovascular diseases (CVD), impaired physical function, activity and increased frailty. Integrating measures of function into community assessments is hindered by the space and time required. The association of function, activity and CVD has not been extensively investigated in COPD. Objectives Explore the potential utility of Time Up and Go (TUG) as a measure of physical function in COPD Assess association of non-invasive measures of haemodynamics to physical function and self-reported activity Explore ambulatory haemodynamics in COPD and controls Methods Subjects with COPD (n=119) and controls (n=58) were recruited. Ethical and governance approvals were obtained. A medical history including falls, spirometry, peripheral and central haemodynamics, self-reported physical activity questionnaires and functional assessments (TUG and six-minute walk distance (6MWD)) were obtained from all subjects. Ambulatory 24-hour haemodynamics including aortic pulse wave velocity (aPWV) and blood pressure were measured in patients (n=20) and controls (n=19). Results TUG mean(SD) was increased in patients 11.9(3.7)s compared to controls 9.5(1.8)s, p < 0.001. In patients, fallers had longer TUG than non-fallers (p=0.02) and a cut-off time of 12s had the highest sensitivity and specificity to detect fallers and non-fallers. Aortic stiffness was not associated to physical function or physical activity, p > 0.05. In the pilot study, significant nocturnal dip in aPWV was seen in controls, p < 0.01, but not in patients, p=0.07. Conclusion TUG could be a useful measure of function and possibly be incorporated into COPD assessment, particularly where time and space are limited. Finally, ambulatory haemodynamic machine, the Mobil-O-Graph, is feasible and offers opportunity to assess 24-hour haemodynamics profile including aPWV as opposed to a one-off measurement.

The chronic infection of the cystic fibrosis (CF) lung with Pseudomonas aeruginosa strongly correlates with critical outcomes. Pseudomonas alkyl-quinolone signal (PQS) is a diffusible cell-density dependent signal controlling the production of virulence determinants. The PQS amount in the CF lung was proportionate to P. aeruginosa colonisation and PQS molecules have been demonstrated to inhibit pro-inflammatory signalling. However, how PQS influence the recognition of P. aeruginosa by the human lung is unknown. The contribution of PQS to the interaction of P. aeruginosa with human bronchial epithelial cells (HBECs) was characterised using a PQS-deficient mutant ΔpqsA in comparison with its isogenic wild type (WT). Although ΔpqsA appeared attenuated, the pathogenesis of WT and ΔpqsA upon infection of HBEC did not differ in bacterial growth, actin and junctional protein degradation, and pro-inflammatory activation. Despite PQS being highly secreted by a CF isolate LESB58, preliminary data showed that LESB58 was less cytotoxic than the laboratory WT. Our results suggest that PQS does not alter P. aeruginosa pathogenicity on HBECs. Idiopathic pulmonary fibrosis (IPF) is characterised with heterogeneous pathological patterns caused by scarring leading to irreversible destruction of lung architecture. Emerging evidence suggests that dysregulated immunological events could cause the failure of tissue-healing. Systemic immune responses of patients with IPF and age- and sex-matched healthy donors were determined by quantifying cytokines produced by peripheral blood mononuclear cells (PBMCs) upon an array of stimuli. The results showed that PBMCs in patients with IPF were less likely to produce IL-17A, IL-10 and IL-13 than healthy controls (OR 0.14-0.3, 95% CI 0.003-0.03). Patients with lower levels of cytokines had a four to six-fold increased risk of death (HR 4.31-6.13, 95% CI 0.0052-0.0176). This study contributes to a better understanding of the role of PQS in P. aeruginosa pathogenesis and identified cytokine production as a novel biomarker in IPF.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease involving both innate and adaptive immune responses. Abnormal numbers of inflammatory cells have been examined in COPD subjects, as well as the effects of cigarette smoking on immune cells and molecules. Killer cells, including CD8+ T cells, NKT-like cells and NK cells, are thought to play a role in the development of COPD through their cytotoxic functions. In this project, we report ex vivo, activation levels of these cell types in COPD patients, as well as effects induced by cigarette smoke extract in vitro. PBMCs were collected from healthy non-smokers (HNS), current healthy smokers (HS), current smokers with COPD (cuS-COPD) and ex-smokers with COPD (exS-COPD). Activation levels of interest and CSE effects on them were analysed by flow cytometry. Killer cells, including CD8+ T cells, NKT-like cells and NK cells, were significantly activated in current smokers with or without COPD compared to healthy non-smokers. Furthermore, KIR (CD158e1) expression was dramatically lower in smokers with or without COPD in comparison with healthy non-smokers. The cytotoxicity of CD8+ T cells from both current smokers and ex-smokers with COPD patients were significantly less than that in healthy volunteers. Also, in vitro, CSE markedly decreased IL-15 treated NK cell activation in current smokers with COPD compared to other three groups. The expression of granzyme B was also significantly inhibited on IL-15 stimulated NK cells when CSE was added. We conclude systemic ex vivo killer cell activation is smoking rather than disease related. Cigrette smoking has immunosuppressive effects on killer cell activation and granzyme B expression in PBMCs from current smokers with COPD.

Colonisation and persistent lung infection by pathogens, mainly Pseudomonas aeruginosa (PA), is an important cause of morbidity and mortality in Cystic Fibrosis patients. PA persists inside the lungs by forming drug-resistant biofilms. Investigation into the behaviour of bacterial cells within these structures and their response to components of the innate immune response will give us an insight into novel approaches to combat infection. The main aim of this thesis was investigate the effect of inflammatory mediators such as cytokines and immune cells on the development of PA biofilms. For this the BioFlux system and PA expressing green fluorescent protein were used to generate biofilms. After incubation with serum, cytokines such as GM-CSF and IFN-γ, changes in the characteristic of the biofilms were analysed. No significant changes on PA biofilms were observed in the presence of the cytokines but human serum was shown to have an inhibitory effect. Incubation of PA biofilms with labelled purified human monocytes and macrophages indicate that macrophages, unlike monocytes attached to PA biofilms. Cell surface receptors expressed by monocytes and macrophages were compared and, as previously described, high mannose receptor (MR) expression was found in macrophages. MR is a carbohydrate binding receptor that plays a crucial role in innate immunity. MR binds mannose-rich carbohydrates through C-type lectin-like domains (CTLD) 4 to 7. Using a recombinant protein containing the mannose binding region of MR (CTLD 4-7-Fc) it was found that MR can directly interact with PA biofilms and that this binding is increased when Psl expression is increased. Furthermore CTLD 4-7-Fc was shown to bind crude extracts of carbohydrates from PA biofilm enriched for the extracellular polysaccharide Psl. We hypothesize that MR expressed by macrophages and dendritic cells might contribute to PA recognition through Psl. Future work will explore the capability of immune mammalian C-type lectins to recognise PA biofilms and purified Psl and elucidate the role of Psl in modulating the recognition of PA by immune cells using PA strains expressing different combinations of extracellular polysaccharides. Our results will shed light on the impact of Psl on the immune recognition of PA, and might open the avenues for the discovery of novel therapeutic targets. Dispersion is the final stage of the biofilm cycle and contributes to dispersal and transmission of pathogens to colonize new sites. Till now not many studies have focused on the role of dispersion in pathogenesis. Thus the present study also focused on the contribution of a dispersal molecule produced during PA biofilm formation termed cis-2-decenoic acid (CDA) and analyse its role in immunomodulation. Previous observations showed that addition of exogenous CDA is able to disperse mature biofilm and inhibit biofilm formation of different gram negative and gram positive bacteria such as PA, E. coli and S. aureus. The present study showed that exogenous addition of CDA is able to modify some aspects of the inflammatory response by modulating production of the pro-inflammatory cytokine TNF-α by human monocytes and macrophages. Thus the present study has identified the potential impact of CDA as an immune modulator which will offer many opportunities for development of drug with potential anti-inflammatory action.

Injury to the bronchial epithelium in respiratory diseases such as asthma and COPD results in the loss of barrier function and an elevated sensitivity to environmental insults. An increased release of the endogenous cannabinoid, anandamide in response to inhalation of allergen in asthmatic patients has been reported. In contrast, previous clinical trial findings suggest anti-inflammatory and broncho-relaxant properties of the phytocannabinoid, ∆9-tetrahydrocannabinol (THC). The aim of this study was, therefore, to determine the effects of endocannabinoids and phytocannabinoids on bronchial epithelial cell permeability and to investigate the mechanisms involved. Calu-3 human bronchial epithelial cells were cultured at air-liquid interface to allow development of tight junctions. Changes in transepithelial electrical resistance (TEER), a reflection of epithelial permeability, were measured at various time points post-treatment. The endogenous cannabinoid anandamide produced a significant reduction in TEER, which was unaffected by cannabinoid receptor antagonists, but attenuated by URB597, an inhibitor of fatty acid amide hydrolase, and by a combination of cyclooxygenase and lipooxygenase blockade. Subsequent immunoblotting data revealed that the expression of tight junction proteins, occludin and ZO-1, were also reduced by anandamide. Inhibition of ERK activation by MEK1/2 inhibitors, PD98059 and U0126, prevented the anandamide-induced reduction in TEER and prevented the reduction in occludin expression. Thus, ERK activation is likely to mediate these effects by altering the expression of tight junction proteins. Treatment with THC prevented TNFα-induced decrease in TEER and increased in paracellular permeability. CB1 and CB2 receptor-like immunoreactivity was found in Calu-3 cells. Subsequent pharmacological blockade of either cannabinoid receptor inhibited the THC effect. In comparison, stimulation of both or either CB1 or CB2 receptors displayed similar effect to that of THC. Western immunoblotting also revealed reproducible decreases in occludin and ZO-1 expression in TNFα-treated cells, whereas cells pre-incubated with THC alone or in combination with TNFα did not alter expression levels. Phosphorylation of myosin-phosphatase target protein at threonine 696 residue by TNFα was attenuated in the presence of THC, indicating the involvement of RhoA/ROCK cascade. Selective stimulation of either cannabinoid receptor in TNFα-treated cells suggests THC-induced inhibitory effect on RhoA/ROCK signalling was mediated through CB2 receptor, and not CB1. In summary, these data suggest that the reduction in transepithelial resistance by anandamide, indicative of increased epithelial permeability, is caused by its metabolites rather than anandamide itself. Inhibition of anandamide degradation might provide a novel approach to treat airway inflammation. Conversely, THC reverses the reduction in transepithelial resistance caused by TNFα, through an effect at CB1 and CB2 receptors. Hence, THC, or perhaps other cannabinoid receptor ligands may have potential therapeutic roles in inflammation-induced changes in airway epithelial cell permeability, such as asthma and COPD.