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Cystic fibrosis (CF) is the most common fatal inherited single gene defect in Caucasian populations. CF lung disease is characterised by infection, inflammation and progressive lung destruction. Lung inflammation is measurable in CF patients even with early disease. Gene therapy offers a theoretical cure for CF lung disease, with large clinical trials now being planned. There is not only a clear need for the development of new therapies in CF but also the means to measure the success of these therapies. One possible approach is to measure biomarkers of airway inflammation non-invasively (in sputum or serum). In this thesis I have employed a number of techniques to measure potential biomarkers in sputum and blood in both cross sectional and serial samples following treatment. Sputum was collected from patients with CF and a number of control groups including Asthma, Bronchiectasis, COPD and healthy controls. SELDI-TOF mass spectrometry was utilised to identify candidate protein biomarkers in sputum. Candidate biomarkers were then identified and compared to established biomarkers by ELISA in sputum. Emission spectroscopy was used to measure metal ions as non-protein biomarkers in sputum. SELDI TOF, ELISA and optical spectroscopy were used to measure biomarkers in CF sputum before and after exacerbation treatment. Calprotectin was also measured in serum before and after exacerbation. SELDI TOF identified calprotectin as a marker of CF lung disease, which highly discriminated CF from control. This could also be measured by ELISA and compared favourably to other inflammatory markers such as Interleukin-8 (IL-8). Emission spectroscopy identified sputum zinc and iron as discriminatory markers of CF. Sputum calprotectin and zinc levels changed significantly following treatment of CF exacerbation. Serum calprotectin also changed significantly and could predict future outcome in these patients. In this thesis I demonstrate the discover}' and application of novel biomarkers of CF lung inflammation. I describe calprotectin (sputum and serum) as useful in the monitoring of exacerbation therapy, with similar findings being displayed for sputum zinc. Further work is now required to fully validate these findings for translation into clinically useful tools.

Background: Non-cystic fibrosis bronchiectasis is a chronic debilitating disease. A perpetual cycle of excessive neutrophilic inflammation and chronic bacterial infection is thought to occur within the permanently damaged and dilated airways. Aim: The aims of this thesis were to explore whether the vicious cycle of infection and inflammation in bronchiectasis can be disrupted, improving the clinical features of the disease and to identify potentially useful clinical markers for monitoring treatment response. Methods: Patients with a radiological diagnosis of bronchiectasis and a clinical history of a daily productive cough, recurrent infective exacerbations and evidence of chronically infected sputum were recruited. The first study was a randomised crossover trial of regular chest physiotherapy in 20 patients assessing whether augmentation of the impaired mucociliary system can improve health related quality of life (HRQL) and the clinical features of bronchiectasis. The second study was a prospective cohort study of 32 infective exacerbations of bronchiectasis managed with two weeks' intravenous antibiotic therapy and assessed the impact of reducing bacterial infection in the airways during acute exacerbations on HRQL, sputum bacteriology, purulence and volume as well as lung function and systemic markers of inflammation. The third study was a randomised crossover trial of regular, long term nebulised gentamicin over 12 months in 57 patients exploring the impact on airways infection and inflammation, lung function, exercise capacity, HRQL and exacerbation frequency. The fourth study sought to identify potentially useful markers of response to chronic treatment strategies for stable disease by analysing markers common to the preceding two interventional studies to assess their robustness and relevance, including sputum bacteriology and purulence, lung function, exercise capacity, systemic inflammatory markers and exacerbation frequency. Results: Augmenting the impaired mucociliary system with regular chest physiotherapy improved HRQL and exercise capacity as well as increasing 24 hour sputum volume. Treatment of acute infective exacerbations improved patients' HRQL and exercise capacity as well as reducing 24 hour sputum volume, improving sputum purulence and decreasing systemic inflammation. Long term regular nebulised gentamicin significantly reduced sputum bacterial density and airways inflammation. Exercise capacity and HRQL improved with treatment and patients suffered fewer exacerbations. Parameters that may be useful in predicting a successful response to long term treatment strategies for clinically stable disease include an improvement of ≥ 50m in the incremental shuttle walk test and eradication of pathogens from the sputum. Conclusion: Augmentation of the impaired mucociliary system, effective treatment of acute infective insults and reducing chronic bacterial infection improves the clinical features of bronchiectasis and suppresses the vicious cycle of infection and inflammation. Potentially useful markers of a successful treatment response in the management of stable disease are exercise capacity and sputum bacterial clearance.

Chronic obstructive pulmonary disease (COPD) has been and remains a major cause of morbidity and mortality across the world. The studies reported in this thesis describe some of the important concepts which have been tested and translated into routine clinical practice in the last 3 decades. We have now clarified the reflex mechanisms underlying persistent cough in COPD, defined the non-specific nature of the sensation of breathlessness in COPD and established that sleep quality is poor in hypoxaemic patients. Secondary polycythaemia is strongly related to carbon monoxide exposure from cigarettes which can also impair exercise tolerance. However the principal reason for exercise limitation in COPD patients is dynamic hyperinflation together with the response of the chest wall muscles to changing lung volume. Defining bronchodilator responsive patients is difficult as the chance of being classified as a responder varies with random fluctuations in baseline FE\A. Expiratory flow limitation at rest is a useful descriptive variable in characterising COPD but is not a predictor of response to bronchodilator drugs. COPD exacerbations are still defined by symptom change which does not always agree with the use of therapy, the commonest outcome reported in clinical trials. However events defined by health care use show a consistent pattern over time and patients who exacerbate often in one year are highly likely to do so in subsequently. Exacerbations are associated with worsening lung mechanics and increased operating lung volume which decreases as the episode resolves. Oral corticosteroids hasten the resolution of these episodes. However hyperglycaemia in patients with respiratory failure is a poor prognostic sign despite non-invasive ventilation. Long-acting inhaled bronchodilators like tiotropium have a sustained bronchodilator effect over the 24 hour day but this does not abolish the normal circadian variation in lung function. Anti-inflammatory therapy with inhaled corticosteroids can reduce exacerbation numbers and improve health status. An effect on mortality has not been conclusively established but seems possible while all treatments so far tested which ameliorate symptoms and reduce exacerbations seem to modify decline in lung function. Another anti-inflammatory agent the PDEIV inhibitor roflumilast has similar effects on exacerbation rate and lung function and may be additive in action. Other non-medical therapy such as heliox can substantially increase exercise performance but are not yet practical for routine use. Rehabilitation, by contrast, can dramatically improve exercise capacity without changing daily activity levels. Despite concerns to the contrary all existing drug treatment is well tolerated and safe. Future studies will need to address earlier intervention not only with smoking cessation -a key intervention of itself- but also with other probably anti-inflammatory therapy which can prevent disease progression and potentially limit the development of co-morbidities. Improvement in patients with more established disease is more likely to follow from the better delivery of the therapy we already possess rather than reversing well established pathology which remains a distant goal at present.

Acute lung injury (ALI) affects a significant proportion of patients requiring critical care and is associated with high morbidity and mortality. Treatment is currently only supportive, with no pharmacological treatment yet shown to definitively improve outcome. There is evidence from murine models of ALI that monocytes play a key role in the development of the neutrophilic lung infiltration characteristic of ALI. Depletion of blood monocytes in mice given intra-tracheal lipopolysaccharide (LPS) significantly reduces pulmonary neutrophil influx, systemic neutrophilia and other markers of lung injury. In humans, monocyte-like cells have been documented in the bronchoalveolar lavage (BAL) fluid of patients with a variety of inflammatory lung conditions, including ALI. This thesis describes novel work performed in healthy human subjects to test whether, in an experimental model of human lung inflammation, depletion of circulating blood monocytes can ameliorate systemic and pulmonary inflammation. LPS inhalation is an established method of modelling ALI in healthy human subjects as it safely and consistently induces mild and self-limiting systemic and pulmonary inflammation. A preliminary study in a group of 12 healthy subjects confirmed the safety and efficacy of LPS inhalation compared to saline placebo. LPS inhalation induced a marked blood neutrophilia together with a rise in body temperature and heart rate and elevated BAL neutrophil and pro-inflammatory cytokine concentrations. This study also used flow cytometry to confirm the presence of pulmonary monocyte-like cells (PMLCs) in BAL fluid, which, although distinct from blood monocytes, could be clearly divided into two separate sub-types according to CD14/CD16 expression. LPS inhalation caused a rise in the number of circulating classical monocytes in blood and an expansion in the CD14++CD16- 'inducible' iPMLC subtype (reminiscent of classical blood monocytes), compared to the CD14++CD16+ 'resident' rPMLC subtype. This may represent transmigration of classical monocytes from blood across the pulmonary endothelium. In humans, mononuclear cell (MNC) leukapheresis provides a readily available method of depleting circulating blood monocytes. A second preliminary study, performed in a separate group of 6 healthy subjects, demonstrated that leukapheresis of four total blood volumes could be safely employed to deplete large numbers of circulating blood monocytes. Active recruitment of monocytes into circulating blood during leukapheresis did, however, limit the reduction in total circulating blood monocyte counts. This study also investigated, for the first time, the potential pulmonary effects of leukapheresis. Despite a relative prominence of iPMLCs in BAL fluid after leukapheresis, there was no evidence of significant neutrophil influx or a clinically important pro-inflammatory effect in the alveolar space. A randomised, double blind, placebo-controlled trial was then performed in a third group of 30 healthy human subjects who all inhaled LPS at baseline. There was no evidence that MNC leukapheresis (depletion group, n=15), compared to a sham procedure (sham group, n=15), attenuated the systemic and pulmonary inflammation induced by LPS inhalation, as measured by: blood neutrophil and plasma C-reactive protein (CRP) levels; by the neutrophil, protein and pro-inflammatory cytokine content of BAL fluid; and by [18F]fluorodeoxyglucose positron emission tomography ([18F]FDG PET)-derived measures of global lung inflammation. MNC leukapheresis temporarily prevented the LPS-induced rise in circulating classical monocytes and was also associated with a small reduction in the estimated numbers of MNCs in BAL fluid. It did not, however, appear to affect the LPS-induced expansion in the iPMLC subtype. Further characterisation of the PMLC subtypes by flow cytometry/sorting and cell culture demonstrated that the iPMLC subtype was more pro-inflammatory but less mature and with a lower proliferation potential than the rPMLC subtype. In summary, this work did not support a role for circulating blood monocytes in the evolution of LPS-induced systemic or pulmonary neutrophilia in man. The rise in circulating levels of classical blood monocytes and the dramatic expansion of pro-inflammatory, immature iPMLCs in BAL fluid after LPS inhalation do, however, suggest that monocytes migrate to the lung and are to some extent involved in the pathogenesis of lung inflammation. Compared to murine methods of monocyte depletion, leukapheresis could not achieve such an extensive or sustained reduction in circulating blood monocyte counts, nor was it likely to have influenced other (specifically patrolling or splenic) monocyte pools. Future work in the drive to find treatments for ALI should therefore investigate the potential of pre-emptive leukapheresis or the efficacy and safety of other methods of human monocyte depletion in experimental lung inflammation.

Background: Bronchiectasis is characterized by chronic cough, sputum production and recurrent chest infections. The pathogenesis is poorly understood but pulmonary pathology shows excess neutrophilic airways inflammation. Accumulating evidence suggests that statins have pleiotropic effects, including modulation of innate and adaptive immune system and anti-inflammatory effects and therefore is a potential novel anti inflammatory therapy for patients with bronchiectasis. Aim: The aim of this thesis was to (i) establish if atorvastatin could break the vicious circle of infection and inflammation in bronchiectasis and (ii) establish the anti inflammatory mechanisms of statins contributing to this. Methods: A RCT was conducted to test the hypothesis that aorvastatin could be a potential anti inflammatory therapy in bronchhiecatsis. In this RCT patients aged 18-79 years were recruited. Patients had clinically significant bronchiectasis, which is patients with cough and sputum production when clinically stable, with two or more chest infections in the preceding year and bronchiectasis confirmed on CT scan of the chest. We excluded: current smokers or ex-smokers of less than 1 year, those with a greater than fifteen pack year history or those with predominant emphysema on CT scan; cystic fibrosis; active allergic bronchopulmonary aspergillosis; active tuberculosis; poorly controlled asthma; pregnancy or breast feeding; known allergy to statins; currently on statins or statin use within 1 year; active malignancy; chronic liver disease; patients on long term oral macrolides; patients chronically colonized with Pseudomonas aeruginosa. Sequence generation was done by block randomization of four, by Tayside pharmaceuticals, NHS Tayside, for 30 patients to receive either atorvastatin 80mg or 30 to receive placebo orally, once daily for 6 months. The placebo (lactose) was not matched to the atorvastatin in appearance. Pharmacy directly dispensed study medications to the patients, hence allocation concealment was maintained at all times to the study investigators. The primary endpoint of this study was a reduction in cough at 6 months compared to baseline as measured by the Leicester Cough Questionnaire (LCQ) score. It is a 19 item self completed quality of life measure of chronic cough which ranges from 3-21, a lower score indicating a more severe cough. The minimum clinically important difference (MCID) is 1.3 Units. The LCQ score is repeatable over 6 months in stable disease. Analysis done was intention to treat. Secondary outcomes included: forced expired volume in one second, forced vital capacity; incremental shuttle walk test; qualitative and quantitative sputum bacteriology; exacerbation frequency; health related quality of life; sputum neutrophil numbers and apoptosis; sputum myeloperoxidase and free elastase activity; sputuminterleukin (IL)-8; systemic inflammation-white cell count, C-reactive protein (CRP) and erythrocyte sedimentation rate, additional systemic inflammatory markers (IL-1β, IL-6, IL-8, IL-10, IL-12p70 and tumor necrosis factor-α) and safety of therapy. Findings: (i) RCT There was evidence of a difference in baseline to 6-month change in LCQ between the treatment groups, with a significant improvement in the statin treated group, with a mean difference 2.2, 95% CI for difference (0.5, 3.9) p=0.01. When analyzed as proportion of improvement in LCQ, in the statin treated group 40% patients had a 1.3 Units or more improvement in the LCQ compared with 17% in the placebo group; difference in proportion 23% (95% CI for difference 1%, 45%), p=0.04. There was significantly increased number of apoptotic airway neutrophils [mean difference of 8.9 (11.7); p=0.04] with a trend towards a decreased total number of neutrophils in the sputum; p=0.09; in statin treated group. Ten (33%) patients had an adverse event in the statin group compared to three (10%) in the placebo, difference in proportion 23% (95% CI for difference 3%, 43%), p=0.02. There were however no serious adverse events. (ii) In vitro studies Statins enhance apoptosis of neutrophils in vitro and this is consequent to reduction in stimuli induced increase in calcium flux. Interpretation (i) In this proof of concept study, six months of atorvastatin improved cough in bronchiectasis. Multi-centered studies are now needed to assess whether long-term statin therapy can reduce exacerbations. (ii) Further studies are needed to establish if statins regulate Ca2+ flux by altering the extracellular or intracellular pathways.

Introduction: Acute respiratory distress syndrome (ARDS) is a major cause of death on intensive care units. The syndrome is characterised by severe hypoxia, widespread atelectasis and decreased lung compliance, much of this impairment due to depletion and inhibition of surfactant. Therapy is supportive. For research purposes, many of the features of ARDS may be mimicked by a saline lavage model of lung injury. Perfluorocarbons have many characteristics similar to natural surfactants and have been used as an experimental means of supporting the injured lung, applied either by special liquid ventilators or as a hybrid technique with conventional gas ventilators known as partial liquid ventilation. Although nebulization is an accepted means of drug delivery, Perfluorocarbons had never been applied by a nebulized route to support the injured lung. Surfactant therapy has also been used to support the lung in the presence of ARDS. Surfactant preparations can be split into two broad categories; artificial surfactants containing no surfactant proteins and natural, but more expensive protein containing surfactants. There may be additional benefits of combining surfactant with Perfluorocarbons. Methods; This study examined the differences between saline lavaged lung injured rabbits, in the following treatment groups; i) control, ii) partial liquid ventilation i.e. poured perfluorocarbon PF 5080; iii) nebulized PF 5080, iv) the artificial surfactant Pumactant used in isolation, v) the natural porcine surfactant Curosurf used in isolation, vi) Pumactant used in combination with partial liquid ventilation and vii) Curosurf used in combination with partial liquid ventilation. The following end points were examined; a) Survival in saline lavaged rabbits to 12 hours, b) Differences in oxygenation between the treatment groups to 6 hours, c) Differences in dynamic compliance between the treatment groups to 6 hours, and static compliance between control, the Pumactant and the Curosurf groups, d) The appearance of computed tomography densities between the control, nebulized and partial liquid ventilation groups in frozen, ex- vivo lung preparations. Results; Survival to twelve hours in this study was greatest in the partial liquid ventilation alone or the combination of Pumactant with partial liquid ventilation. Oxygenation was improved by partial liquid ventilation, the natural surfactant Curosurf, or the combination of either Pumactant, or Curosurf with partial liquid ventilation. Dynamic compliance improved after partial liquid ventilation, Curosurf in isolation or the combination of Curosurf with partial liquid ventilation. Static compliance improved after treatment with Curosurf but not after treatment with Pumactant. There was a significant difference in density distributions in the CT scanning studies between the partial liquid ventilation and nebulized perfluorocarbon, and the control and partial liquid ventilation groups, but not between control and the nebulized groups implying little perfluorocarbon is delivered to the lungs by this route. Comments; In summary PF 5080 can be used for partial liquid ventilation in this model of lung injury to improve survival, oxygenation and lung mechanics. Using this method, nebulization of PF 5080 cannot be supported as it seems to have little effect on these end-points and may not be delivered in any significant amount to the respiratory tract. Curosurf is superior to Pumactant in improving oxygenation, and lung mechanics in this model of acute lung injury. There seems to be little difference between Curosurf and poured PF 5080 in terms of these endpoints. Whether partial liquid ventilation becomes more widespread in the support of the injured lung will depend on further research applications including means of application and trials in humans.

Introduction and Objectives. Pulmonary and cardiovascular responses in healthy volunteers, patients with chronic obstructive pulmonary disease (COPD) or ischaemic heart disease (IHD) were investigated following exposure to the high levels of diesel pollution on London’s Oxford Street. Methods. Using a cross-over design 40 healthy volunteers, 40 patients with COPD and 39 patients with IHD walked along Oxford Street (diesel only traffic) and, on a separate occasion, in Hyde Park (traffic free) for two hours. Cardio-respiratory measurements were performed at baseline and during and after each exposure, alongside personal particulate exposure measurements. Findings. Pulse wave velocity (PWV), a measure of arterial stiffness, increased in healthy subjects 24 hours after Oxford Street exposures started compared to Hyde Park (0.6m/s vs -0.4m/s, co-efficient=0.43, 95% CI 0.25-0.61, p < 0.001). This phenomenon was also seen in COPD subjects 24 hours after exposures started (0.4m/s vs -0.4m/s), with the ‘Oxford Street’ effect statistically significant (co-efficient=0.50, 95% CI 0.33-0.68, p < 0.001). There were no significant changes in PWV seen in the IHD subjects. Healthy volunteers had a 1.6% increase in FEV1 five hours after the start of exposure on Oxford Street, compared to a 7.2% increase in Hyde Park. Similar changes were noted six and 24 hours after the exposure started (co-efficient= -2.13, 95% CI -3.23 to -1.02), p < 0.001). FEV1 in COPD subjects dropped by 2.2% one hour after the exposure started on Oxford Street (co-efficient=-1.36, 95% CI -2.24 to -0.47), p < 0.003). Measurements of impulse oscillometry demonstrated increased airway resistance in subjects with COPD at 5 Hz (R5) of 0.05 kPa/l/s four hours after the exposure started on Oxford Street (co-efficient=-0.03, 95% CI 0.01 to 0.04), p=0.001), and at 20 Hz of 0.02 kPa/l/s 24 hours after exposure (co-efficient=0.017, 95% CI 0.01 to 0.027), p < 0.001). The IHD subjects had significant increases in airways resistance at R5 four hours and 24 hours after the exposure started on Oxford Street (0.07kPa/l/s and 0.01kPa/l/s respectively, co-efficient=0.03, 95% CI 0.01 to -0.06, p=0.015). Conclusions. Vascular dysfunction was observed in all the volunteer groups following exposures on Oxford Street. Volunteers with COPD were observed to have increased airflow obstruction.