Neutrophil transit through the pulmonary circulation is delayed in comparison to the transit of erythrocytes. During inhalation of cigarette smoke, this delay or sequestration of neutrophils, which occurs in normal lungs, was found to be enhanced. This could facilitate neutrophil emigration into the airspace or activation of sequestered neutrophils could result in an elastase burden in the pulmonary vasculature. Hence, studies of the mechanism of the enhanced neutrophil sequestration during smoking would improve our knowledge and may lead to therapeutic interventions for smoke-induced lung diseases. The intravascular pulmonary sequestration of neutrophils is influenced by haemodynamic forces, the cell's ability to deform, and by an increase in adhesion between neutrophils and endothelial cells. This thesis has concentrated on the influence of cell deformability on neutrophil sequestration in the lungs and the effects of cigarette smoking. An <I>in vitro </I>measurement of cell deformability was compared to the <I>in vivo</I> kinetics of neutrophils in the lungs of man, to reveal a strong correlation between the first pass sequestration <I>in vivo</I> and <I>in vivo</I> cell deformability. Exposure of isolated neutrophils to the vapour phase of cigarette smoke <I>in vitro</I> caused a reduction in cell deformability. However, recovery of cell deformability was observed with time. The functional behaviour of smoke exposed neutrophils was also adversely affected. The mechanism for the reduction in cell deformability an exposure to cigarette smoke appeared to be due to the oxidant effect of smoke on the cell cytoskeleton. Moveover, in confirmation, a reduction in leucocyte deformability was observed in arterial but not venous blood, by measuring the filterability of diluted whole blood, following <I>in vivo </I>cigarette smoking.

Emphysema is a degenerative lung disease which, it is suggested, results probably from repeated periods of proteinase:antiproteinase imbalance during which excess of enzyme attacks the extracellular matrix. Of the many enzymes produced by inflammatory cells human neutrophil elastase (HNE) is thought to be the major offending enzyme. It attacks elastin, which is responsible for the elastic recoil of the lung. If emphysema was simply a result of the destruction of elastin by HNE then degradation products of elastin would inevitably be present, at least transiently, in the serum of patients. The aim of this project was to separate and characterise the soluble peptides resulting from the digestion of elastin with HNE and/or human neutrophil cathepsin G (HNCG), another neutrophilic enzyme, which is primarily bactericidal and, to determine if any of the peptides were characteristic of digestion by one enzyme or combination of enzymes. HNE and HNCG were isolated from purulent sputum, and elastin was isolated by two methods from post-mortem lungs. The digestion of the elastin by the enzymes was followed by measuring the amino groups liberated during the course of the digestion. A method was developed for the measurement of the insoluble as well as the soluble products of digestion. Initially, the amounts of soluble and insoluble products were similar, but the amount of soluble products soon exceeded the amount of insoluble products. The soluble products of digestion were separated by reverse-phase chromatography. The peptides separated into two groups (A and B), regardless of which enzyme was involved in the initial digestion. Both groups were heterogeneous mixtures of peptides. Filtration experiments and amino acid analysis showed that the groups of peptides differed in size and composition.

Biopharmaceuticals are finding wide applications in the management of diverse disease conditions. Pulmonary delivery of proteins may constitute an effective and efficient non-invasive alternative to parenteral delivery, which is currently the main route of administration of biopharmaceutical drugs. A particular area, in which pulmonary delivery of peptides and proteins may find ready application, is in the local delivery of antimicrobial peptides and proteins to the airway, a measure that could potentially bring about improvements to currently available antipseudomonal therapies. This thesis has therefore sought to develop inhalable antimicrobial proteins in combination with antibiotics that have particularly good antimicrobial activity against Pseudomonas aeruginosa infections in the respiratory tract of people with cystic fibrosis (CF). Through process optimisation, a suitable spray drying method was developed and used for the preparation of active, inhalable dry powder formulations of the antimicrobial protein, lactoferrin, and aminoglycosides (tobramycin and gentamicin). The physicochemical properties, aerosolisation performance and the antibacterial properties of the various spray-dried formulations were assessed. In addition, a relevant in vitro cellular model was employed to investigate the potential cytotoxic and pro-inflammatory effects of the various formulations on four bronchial human epithelial cells together with their effectiveness at reducing bacterial colonies when administered on to biofilm co-cultured on the epithelial cells. It was found that following spray drying the particles obtained were mostly spherical, amorphous and possessed suitable aerosolisation characteristics. The various spray-dried antimicrobial proteins (lactoferrin or apo lactoferrin) and co-spray dried combinations of the proteins and aminoglycosides were found to exhibit bactericidal activity against planktonic and biofilms of P. aeruginosa. In general, the spray drying process was found not to significantly affect the antimicrobial activities of the protein. Treatment of the different bronchial epithelial cell lines with the antimicrobial formulations showed that the various formulations were non-toxic and that the co-spray dried combinations significantly reduced established P. aeruginosa biofilms on the four bronchial epithelial cells. Overall, the results from this thesis demonstrates that spray drying could potentially be employed to prepare inhalable antimicrobial agents comprised of proteins and antibiotics. These new combinations of proteins and aminoglycosides has promising applications in the management of P. aeruginosa in the airway of cystic fibrosis patients.

Chronic obstructive pulmonary disease (COPD) is an inflammatory condition of the lung caused by an abnormal response to particles and noxious gases, primarily cigarette smoke. Patients suffer daily symptoms and can have episodes of worsening symptoms termed acute exacerbations. Exacerbations are associated with impaired quality of life, faster lung function decline, higher mortality and increased risk of hospitalisation. The aetiology of COPD exacerbations is controversial; however respiratory viral and bacterial infections are an important feature of exacerbations. This study utilised real-time qPCR to measure prevalence and load of human rhinovirus (HRV) in stable COPD and during the time-course of naturally occurring exacerbations and their recovery. HRV was assessed in association with upper respiratory tract (URT) symptoms namely cold symptoms and sore throats, secondary bacterial infection, patient reported outcomes and exacerbation frequency. Additionally, respiratory syncytial virus (RSV) was semi-quantitatively examined in stable COPD and at exacerbation. The original contribution of this work to the field is that HRV prevalence and load are highest at exacerbation presentation and decrease during recovery. HRV load is higher in the presence of URT symptoms compared to the load without, and the load remains higher for longer with both symptoms compared to only one. This study described novel evidence for the development of secondary bacterial infection after HRV infection in natural exacerbations, and demonstrated that HRV infection is associated with patient reported outcomes. Patients with HRV had higher exacerbation frequencies compared to those without HRV. RSV prevalence did not change significantly between stable COPD and exacerbation. The findings from this thesis have important implications in terms of exacerbation therapy. The evidence provided may allow appropriate targeting of therapeutic interventions therefore reducing exacerbation severity and frequency. These findings emphasise the importance of rapid development of therapeutic targets for the prevention and treatment of HRV infection in COPD patients.

CRTh2 (DP2) is implicated in the pathogenesis of asthma; however, currently there is a lack of data describing the protein expression of CRTh2 in bronchial biopsies in asthma. This has limited the cell types that CRTh2 function has been explored within. A thorough understanding of CRTh2 expression within the airways and whether changes in receptor expression correlates with disease severity, may aid in the design of future CRTh2 antagonist clinical studies. This study aimed to investigate the expression of CRTh2 expression in bronchial biopsies of subjects with asthma and healthy controls. The novel finding that CRTh2 was expressed on bronchial epithelial cells in asthma prompted further investigation into the expression and activation of CRTh2 on bronchial epithelial cells in vitro, using the selective CRTh2 agonist 13, 14-dihydro-15-keto prostaglandin D2 (DK-PGD2) and the CRTh2 selective antagonist AZD6430. This study is the first to describe differential CRTh2 expression within bronchial tissue in asthma compared to healthy controls. The number of sub-mucosal CRTh2+ cells was found to be increased in asthma compared to healthy controls. CRTh2 was found to be expressed on the bronchial epithelium and its expression was decreased in asthma compared to healthy controls with similar differences observed in vitro by primary epithelial cells. Squamous metaplasia of the bronchial epithelium was increased in asthma and related to decreased CRTh2 expression. DK-PGD2 promoted epithelial cell migration, and in air-liquid interface cultures increased the number of MUC5AC+ and involucrin+ cells, which were blocked with the CRTh2 antagonist, AZD6430. This study describes the novel findings that CRTh2 is expressed by the bronchial epithelium in both health and asthma, and its activation drives epithelial differentiation. These data suggests that CRTh2 could contribute to airway remodelling in asthma and this information may contribute to the understanding of the effects of CRTh2 antagonists in asthmatic patients.

Introduction: It is hypothesized that bacteria are important in the pathogenesis of COPD and exacerbations. Most bacteriological research in COPD has utilised culture based methods. Novel molecular approaches enable detailed evaluation of the airway microbiome that may better inform the role of bacteria in COPD. This project aimed to characterise the microbial community in COPD at stable state and during acute exacerbations through assessment of serial sputa at stable (S), exacerbation (E), follow up (F) and recovery (R) visits. Methods: Sputum from 145 clinical trial COPD patients was collected at multiple stable visits and at each exacerbation (E, F and R visit) over 12 months. Real-time quantitative PCR (qPCR) was performed on sputum DNA using universal 16S gene primers and specific gene targets to quantify total bacterial load and the specific pathogens H. influenzae, S. pneumoniae, M. catarrhalis and S. aureus. In a subgroup of 30 exacerbating patients, 454 high-throughput pyrosequencing of 16S rDNA amplicons was performed at each of the 4 visits. Results: There was no significant difference in total bacterial load or any specific pathogen between longitudinal stable and exacerbation samples. 454 pyrosequencing identified Proteobacteria and Firmicutes to be the dominant groups contributing >80% of the sequence reads at phylum level. Haemophilus, Moraxella and Streptococcus were the dominant groups at genus level. No significant within-subject change in the microbial community was observed across visits. Cluster analysis, based on the ratio of Proteobacteria to Firmicutes (P:F) characterised three subgroups. The high P:F subgroup was characterised by a significant increase in P:F from S to E visits, associated with raised blood CRP and sputum IL-1β levels, suggesting a role for bacteria in exacerbation pathogenesis for this subgroup. Conclusions: Molecular profiling identifies heterogeneity in the airway microbiome of COPD patients, with a role for bacteria suggested in a subgroup.

Vascular endothelial growth factor (VEGF) is a potent mitogenic, angiogenic and permeability factor that has been associated with the development of lung disease. The VEGF gene undergoes alternate splicing of exon 8 to produce two isoform families with differing functional effects. VEGF16sa is the most biologically active member of the VEGFxxxa family. One member of the VEGFxxxb family, VEGF16Sb, has "inhibitory" effects on VEGF16sa induced endothelial proliferation, migration and permeability. This study is based upon the hypothesis that these isoforms utilise differing signalling pathways, identification of which would enable manipulation of their functional effects. To investigate this hypothesis, mRNA and protein expression of VEGF receptors and coreceptors in human pulmonary microvascular endothelial cells (HPMEC) were compared to those of systemic human umbilical vein endothelial cells (HUVEC). Potential downstream signalling pathways and mechanisms underlying VEGF165a and VEGF165b induced changes in cell permeability were investigated using signalling inhibitors and cellular distribution of the cell-junctions proteins VE-cadherin and ZO-l. Finally, PCR array profiles were utilised to screen potential differences in gene expression downstream ofVEGF and nitric oxide. The work presented shows that HPMEC and HUVEC cells differ in basal expression ofVEGF receptors and co-receptors, and that they are modified in response to chronic stimulation with VEGF isoforms. Cell signalling pathways downstream of VEGF-R2 differ between VEGF16sa and 16Sb and between the cell types. Three different methods (Endohm, ECIS and FITC-BSA passage) showed that VEGF16sa induced an increase and VEGF16Sb a decrease, in permeability for both HPMEC and HUVEC. L-NAME, L-NIO and PI3K (AKT leNOS pathways) inhibitors, blocked both VEGF isoforms suggesting no differential signalling in this pathway. Observation of changes in VE-cadherin and ZO-l expression pattern at cell junctions after VEGF16sa stimulation but not after VEGF16Sb suggested that this was a possible mechanism involved in the regulation of HPMEC and HUVEC permeability. PCR profiling array suggested some potential differential gene expression following VEGF16sa and VEGF16Sb stimulation. Understanding the signalling pathways utilised by VEGF isoforms to regulate lung biology could enable us to preferentially induce specific beneficial effects in the lung such as epithelial mitosis, whilst inhibiting detrimental effects such as increased permeability.

Introduction Malignant pleural disease is a growing clinical problem and often results in substantial breathlessness for patients due to the accumulation of malignant pleural effusion. Dedicated pleural services are becoming more prevalent and a wider selection of treatment strategies are now available. This thesis evaluates a number of different aspects relating to the prognostication and treatment of patients with malignant pleural disease through a series of 4 studies. Methods The first study uses data from three, international, prospectively collected databases of patients with malignant pleural effusion to evaluate factors that predict patients' survival. From this data, the LENT prognostic score is developed and validated to assist clinicians in risk stratifying patients and thereby helping them to select appropriate treatment strategies. The second study is a systematic review of the literature, evaluating all the published randomised controlled trial (RCT) data on the management of malignant pleural effusion using network meta-analysis. These results highlight the efficacy of talc poudrage in obtaining a pleurodesis, but also emphasise the heterogeneity of the available evidence and the paucity of robust data on symptom based outcomes and adverse effects. The Zoledronic Acid (ZA) Trial is a pilot, RCT evaluating whether intravenous ZA is an effective potential treatment for MPE. In this small, diverse cohort, no significant difference was seen in the radiology, biomarkers or symptoms of the ZA group compared with those who received placebo. Finally, the SMART trial is a multi-centre RCT, evaluating the role of prophylactic radiotherapy in mesothelioma. 203 patients have been recruited to the study, which is currently in follow up and the results are awaited in winter 2015. Discussion This thesis adds to our understanding regarding the prognostication and management of patients with malignant pleural disease. The data will help to inform clinicians and future research studies regarding the optimal management of these patients

Patients with difficult to manage asthma and chronic cough are commonly seen by respiratory physicians in the NHS. This thesis describes three randomised trials which explore new treatment options for these difficult groups. Non-invasive markers of airway inflammation and function were measured before each trial to help determine likely responders. In the first study, 30 patients with asthma and eosinophilic inflammation were given two weeks of prednisolone and then randomised to receive either ciclesonide 360mcg or placebo twice daily for 8 weeks. Though the between- group differences were not significant several patients had changed their usual maintenance dose of prednisolone during the trial. When these patients were removed from the analysis there was a significant improvement sputum eosinophils with ciclesonide. There was no significant change in the marker of small airway inflammation, so it is possible that this effect was due to a general reduction in airway inflammation from the higher dose of inhaled steroids, rather than specifically targeting the small airways. In the second study, 28 patients with refractory asthma were given azithromycin 250mg or placebo three times weekly for six weeks in a randomised, cross-over design. Though significant improvements in airway hyper-responsiveness, asthma control and sputum neutrophils were seen with azithromycin, these changes were not significant when compared to placebo.

Background: Hyperglycaemia predicts adverse outcomes in patients admitted to hospital for exacerbations of chronic obstructive pulmonary disease (COPD). Metformin promotes euglycaemia but not hypoglycaemia, making it suitable for use in a pre-emptive anti-hyperglycaemic strategy. Aims: To investigate the effects of met form in in COPD exacerbations, in particular to confirm or refute a glucose-lowering effect. Ultimately, to determine whether a larger study powered for patient-centred outcomes is justified. Methods: First, a retrospective analysis of the association between metformin, lactate concentration and mortality in diabetic patients with COPD exacerbations. Second, a randomised, double-blind, placebo-controlled trial testing metformin in patients hospitalised for COPD exacerbations. Participants were assigned in a 2:1 ratio to 1 month of treatment with metformin, escalated rapidly to 2 g/day, or placebo. The primary endpoint was mean in-hospital blood glucose concentration, in respect of which 48 participants would provide 90% power to detect a 1.5- mmol/L difference. Secondary end points included other markers of glycaemia, inflammation, recovery, and safety and tolerability. Results: Among the 130 patients in the retrospective study, 51 (39%) were taking metformin. Metformin was associated with a slightly higher lactate concentration (median difference 0.40 mmol/L, 95% confidence interval [Cl] 0.10-0.60), but lower all-cause mortality (hazard ratio 0.57; Cl 0.35-0.94). In the clinical trial, 52 participants were randomised to metformin or placebo (allocation ratio 1.9:1). The mean (SO) blood glucose concentration was 7.1 (0.9) mmoljL in the metformin group and 8.0 (3.3) mmol/L in the placebo group (difference -0.9 mmoljL, Cl -2.1 to +0.3; P=0.273). There were no differences in the secondary endpoints. Adverse reactions, particularly gastrointestinal effects, were more common in metformin-treated participants. Conclusion: In patients hospitalised for COPO exacerbations, pre-emptive metformin therapy does not significantly lower blood glucose concentration. On this basis, a larger trial testing the effects of metformin on patient-centred outcomes in this setting is unwarranted.