Asthma is a complex and heterogeneous disorder, comprising domains of pathology (airway inflammation), physiology (disordered airway function) and clinical expression (symptoms and exacerbations) that are variably related. Within this network, the role of eosinophils remains uncertain. This thesis explores further the relationship between eosinophilic inflammation and clinical asthma by: 1. using multivariate statistical techniques to characterise and classify relationships between individual domains in the asthma population; 2. investigating clinical outcomes with mepolizumab (anti-IL 5) in a randomised placebo controlled trial for 12 months, in subjects with refractory eosinophilic asthma. In the first study I have used factor analysis techniques to formulate statistically independent domains of the clinical phenotype. These have been entered into a cluster analysis algorithm to identify subgroups that define a classification model based on expression patterns and composite relationships across the asthma domains. By applying this independently to populations of Primary and Secondary Care asthma, I have identified two secondary care predominant clusters, characterised by discordance between asthma symptoms and eosinophilic airway inflammation. I have shown that discordant clusters derive greatest clinical benefit with a management strategy directed at maintaining a normal sputum eosinophil count, supporting its implementation in secondary care. More specifically, I report a 10-fold reduction in severe exacerbations with inflammation guided therapy in discordant eosinophil-predominant asthma, supporting a specific role for eosinophils in these events. In the second study, I report a significant reduction in severe exacerbations with mepolizumab therapy, but no effect on other clinical outcome measures. These findings support a specific effector role for eosinophils in the pathogenesis of severe exacerbations; and dissociation of this endpoint and other clinical outcome measures. Finally, I report a 12 month washout analysis of mepolizumab treated subjects in which a significant increase in severe exacerbation frequency is observed that is temporally preceded by rising sputum eosinophils.

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Exhaled breath condensate (EBC) collection, is a non-invasive method of measuring airways inflammation. This thesis records the development of a portable and reusable EBC collection system and its use in health and disease to determine ranges of EBC nitrate and nitrite (NOx) and glutathione (GSH) concentration; additionally the effect of direct and ambient air pollution exposure on NOx and GSH in EBC and nasal lavage NALF) was examined. Preliminary experiments revealed a collection time of 15 minutes with a system employing Teflon® tubes provided mean EBC volumes of 1.94±0.58m1. EBC NOx found in >90% of samples, was reproducible within healthy subjects over 6 collections; whilst EBC GSH was consistently below the limit of detection. EBC NOx levels were higher in asthma and COPD, suggestive of increased airways inflammation when compared to healthy subjects (8.1±7.7;9.0±7.5μmoVL respectively vs. 4.8±2.3μmoVL), whilst salbutamol inhalation in-between EBC collections caused a decrease in EBC NOx in COPD but not in asthma. Exposure to 200ppb sulphur dioxide (SO2) in healthy subjects elicited an immediate oxidative stress response evident by depletion of GSH in NALF which was absent in coronary artery disease (CAD); whilst an increase in NALF NOx was seen at 4 hours post SO2 in the CAD group only. Evidence for lower airways inflammation was supported by an increase in EBC NOx in both groups at 24 hours post SO2. Exposure to carbon particles failed to elicit a response in either group. Chronic lung disease patients exposed to ambient levels of pollution at homes in Athens, Amsterdam, Helsinki and Birmingham, displayed increases in EBC NOx in line with increases in pollution indices; in particular with PMIO and coarse particles measured at a central site and PM2.5 inside the home, particularly in subjects from Athens, where EBC NOx was greater. EBC was reliably collected in this thesis and changes in the inflammatory marker NOx may be detected in response to oxidative stress in the airways due to disease, drug administration and direct and ambient air pollution exposure

Introduction: Varying methods of cough quantification have been used to describe cough frequency, although there is no consensus which method best relates to subjective rating of cough. With increased availability of semi-automated cough recording devices, the capability to carryout anti-tussive studies is greatly increased. However, there is no data available describing the magnitude of change in cough frequency necessary to provide therapeutic benefit. Female patients are over represented in specialist cough clinics. There is little data describing pulmonary function and airway inflammation in phenotyped patients and how these measures may relate to objectively measured cough. Airway inflammation isa common feature of chronic cough, regardless of the trigger for cough. The repetitive mechanical insult of the act of coughing may be responsible for the presence ofthis inflammation. Methods: 100 patients with unexplained chronic cough under went full diagnostic testing in order to determine potential triggers for cough. Procedures included pulmonary function testing; (spirometry, eND, EBC pH, BHR, Cough challenge, induced sputum, objective and subjective cough monitoring), Bronchoscopy, 24 hour impedance monitoring with simultaneous cough monitoring, Gastroscopy and ENT. A subset of cough recordings were quantified in cough sounds, cough seconds and cough epochs to determine the best way to quantify cough. 20 healthy volunteers performed voluntary coughing manoeuvres to determine the acute effects of coughing on airway function and inflammation. Results: Cough sounds and seconds correlate moderately with subjective and QDL . methods. Patients reported a reduced cough frequency whilst undergoing impedance testing; a reduction of33% was seen although patients were unable to quantify the scale of change. Female patients coughed substantially more than male patients, with the largest difference seen at night. Cough frequency was predicted by gender, C5 and age. Cough frequency was not related to a specific trigger for cough. Reduced small airway flows were seen that were independent of BHR. Prominent airway neutrophilia was seen regardless of the trigger for cough. Acute changes in eND and EBC were seen after short periods ofvoluntary coughing. Sputum inflammatory mediator levels showed huge variability and did not change as a result of coughing. Bronchodilation ofthe small airways was also seen after coughing. Conclusion: The use of objective cough monitoring has enabled me for the first time to describe the most appropriate current methods of cough quantification and to demonstrate the magnitude ofreduction in cough frequency that is appreciated by -patients.-I-havereportedimportant significant genderdifferences in cough frequency and shown that cough frequency is predicted not only by gender but also age and cough reflex sensitivity showing important implications for underlying mechanisms of chronic cough. I have also shown for the first time that pulmonary function measures are reduced in patients without indication of asthma. The mechanical act of coughing causes acute bronchodilation of small airways and reduces eND and EBC pH.

The funding for this research project was kindly provided by the Medical Research Council (MRC) and the British Thoracic Society (BTS)Intracellular Ca2+ homeostasis and handling were investigated in passaged human airway smooth muscle, hASM, cells from asthma and normal donors. Temporal changes in fluorescence of Ca2+-sensitive indicator fura-2 loaded into quiescent sub-confluent hASM cells were monitored using epifluorescence video microscopy. Spontaneous amplitude changes in basal fluorescence of temporal waveforms, or Ca2+ oscillations, were measured. Also, spectral analysis using the FFT transform generated a Ca2+ oscillation dominant frequency (CODF) variable. Neither amplitude nor CODF were significantly different in asthma compared to normal hASM cell donors. However, there was a significant difference (P<0.0001) between CODF in airflow obstruction (AFO), defined as FEV1/FVC<70% and FEV1< 80%, and non-AFO donors, making CODF a strong phenotypic predictor of AFO. hASM cell Ca2+ handling was investigated by Ca2+ uncaging using confocal microscopy and by bradykinin stimulation using epifluorescence microscopy. Basal Ca2+ level, Ca2+ handling exponential decay rate constants (K), SERCA activity and expression, and SOCE after a SR Ca2+-store depletion event, all demonstrated that Ca2+ handling was not significantly different between hASM cells from asthma or normal donors. There was no correlation between FEV1 and K, however there was an emerging correlation between FEV1/FVC and K for bradykinin. The postulate that Ca2+ homeostasis and handling are intrinsically dysfunctional in hASM cells from asthma compared to normal donors is ergo not supported by these data. Caffeine was found to decrease basal Ca2+ and inhibit Ca2+ oscillations in hASM cells. Future work using freshly dispersed hASM cells is required to understand in vivo Ca2+ dynamics using the methods described in this thesis. Since CODF correlates with FEV1, pattern recognition of Ca2+ oscillation frequency spectra has the potential to help define clinical asthma phenotypes. Inevitably, a post-genomic approach to comparative protein expression in asthma and normal hASM cell donors will accelerate understanding of Ca2+ dynamics.

Asthma is a complex disorder with environmental and genetic triggers. These have been the subject of extensive study but progress in establishing reliable genetic determinants of this disease have met with only limited success. In this thesis I report on how the asthma phenotype in childhood varies within different populations of asthmatic children and how this might influence the results of our studies and those of other authors. I then critically review each component of the asthma phenotype and discuss its usefulness as· a marker of the disease within the population studied. I then go on to determine genetic associations between individual and parental genotype and phenotypic expression within children using a structured candidate gene approach. Following this approach it is evident that parts of the asthma phenotype, specifically airway hyperresponsiveness as measured by methacholine challenge varies consid~rably between different regional asthma centres.The reasons for this variation are unknown but limit the usefulness of this measure as an endpoint within our cohort 'of children. Subsequent detailed examination of other potential markers of- the asthma phenotype include an asthma severity score based on international guidelines, lung function measurements, total serum immunoglobulin E levels and the results of skin prick tests.

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