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Allergic conditions affect increasing numbers of the population, are associated with considerable morbidity and in their most severe forms can be life-threatening. A prominent feature in allergic conditions is the activation of mast cells and basophils by allergen resulting in the release of inflammatory mediators. A unique product of basophils is basogranulin, a protein stored and released from the secretory granules on cell activation. Our aim has been to develop new assays for assessing basophil activation based on basogranulin measurements, and applying these assays for measuring basophil sensitivity in samples from allergic patients. In addition, we have investigated basogranulin in saliva and BAL fluid as a marker for basophil activation in vivo. Basophils stimulated with fmlp, anti-IgE and grass pollen in vitro and the basogranulin released into supernatants was quantified by dot blotting. In addition, flow cytometric assays were developed for measuring alterations of intracellular and surface basogranulin expression following basophil activation in vitro with fmlp, anti-IgE anti-FcɛRI or specific allergens. There was an apparent depletion in intracellular basogranulin stores in activated basophils when compared with that in non-stimulated basophils. The depletion of intracellular basogranulin was inversely associated with increased expression of CD63. Surface basogranulin expression was barely detected in nonstimulated basophils but was increased following stimulation. Membrane expression of basogranulin was correlated with that of CD63 in nonstimulated basophils (0.829, P< 0.005, n=10), and in basophils stimulated with anti-IgE (r=0.877, P< 0.001, n=51), anti-FcɛRI (r=0.680, P< 0.0001, n=20), or fmlp (r=0.914, P< ii 0.0001, n=9). When basophils were stimulated with extracts of various allergens including D. pteronyssinus, D. farinae, crab, shrimp and oyster increased basogranulin expression wasobserved in cases for where this was not detected for CD63. Alterations in intracellular and surface basogranulin expression were also visualised by confocal microscopy, and by this technique considerable heterogeneity in marker expression was observed between individual cells. Basogranulin could be detected in saliva samples of peanut allergic children, suggesting a potential role in assessing the contribution of basophils in clinical disease. In BAL fluid from patients with atopic asthma basogranulin levels were higher than that from healthy subjects, but not from those with severe asthma. Basogranulin measurement within basophils, on the cell membrane and in biological fluids represent novel approaches for assessing basophil activation and for establishing the contribution of basophils in clinical disease. They show promise as new means for the diagnosis of allergic sensitivity and confirmation of allergic reactions mediated by basophils.

Asthma is a complex heterogeneous disease influenced by many factors, such as genetic background and environmental exposures. This suggests the potential for geneenvironment interactions in which the genotype of an individual is involved in complex interactions with environmental exposures. Of the many asthma susceptibility genes, A Disintegrin And Metalloproteinase 33 (ADAM33 ) has been linked to asthma and airway hyperresponsiveness (AHR), as well as impaired lung function in early life. Several epidemiological studies have shown that maternal asthma and allergic disease lead to an increased prevalence of AHR and asthma in their offspring. Therefore, it was hypothesised that in utero gene-environment interactions between ADAM33 and maternal allergic airway inflammation lead to increased AHR of the offspring post partum. To investigate this hypothesis, the first objective was to establish a mouse model of allergic airway inflammation during pregnancy. Parents heterozygous for Adam33 (Adam33+/-) were used to allow for wildtype (WT, Adam33+/+) and Adam33 -knock out (KO, Adam33-/-) offspring in the same litter. The second objective was to assess the impact of maternal airway inflammation and Adam33 genotype on AHR in these offspring 4 weeks post partum. The third objective was to investigate the underlying mechanisms of this effect by studying the lungs post partum and in utero to identify changes that can contribute to gene-environment interactions with Adam33. It was found that house dust mite challenge of pregnant dams led to a strong eosinophilic airway inflammation, but this did not have any substantial effect on litter size or off- spring weight. However, at 4 weeks of age, WT offspring of HDM challenged dams exhibited significantly increased AHR in response to methacholine compared to WT offspring of saline challenged control mothers, whereas KO littermates of HDM challenged dams were protected. Analysis of the developing lung on ED17.5 by RNA sequencing revealed that loss of Adam33 caused a defect in muscle development and contraction, as well as an impairment of calcium and cGMP-PKG signalling, two pathways involved in the mediation of muscle contraction. The hyporesponsive phenotype of Adam33 -KO airway smooth muscle (ASM) was confirmed 4 weeks post partum by using a thromboxane-analogue in precision-cut lung slices. It was found that the AHR in WT offspring of HDM challenged dams at 4 weeks was caused by an increased expression of the muscarinic M1 receptor (CHRM1), which caused an enhancement of vagal reflexes mediating stronger airway contraction. While KO offspring of HDM challenged mothers showed a similar increase in CHRM1, failure to observe increased AHR was due to hyporesponsive ASM as previously observed; this counteracted the increased vagal reflexes and thus prevented AHR. These data demonstrate for the first time that maternal allergic airway inflammation during pregnancy leads to AHR in allergen-naive offspring 4 weeks post partum. Furthermore, the data identify a role for Adam33 during muscle development in utero, and show that loss of Adam33 can counteract the increased airway contractility mediated by maternal allergy, so protecting against AHR. These studies highlight the importance of gene and environment interactions in the development of asthma in early life and point to potential approaches for future therapeutic intervention.

Pulmonary diseases has long suffered from a lack of clinical biomarkers for disease diagnosis, prognostic prediction and treatment response. Pulmonary surfactant is critically important for optimal lung function. While altered surfactant function, concentration and composition have been reported in patients with various lung diseases, these changes have also yet to be translated into clinical practice. An alternative approach to measurement at a single time point is to quantify time course kinetics of substrate fluxes through metabolic pathways. Consequently, in this thesis, incorporation of the stable isotope-labelled substrate deuterated methyl choline chloride (methylD9choline) into the major surfactant phospholipid phosphatidylcholine (PC), combined with electrospray ionization tandem mass spectrometry (ESI MS/MS), was employed to monitor the metabolic flux of surfactant phospholipid synthesis in vivo. Time series PC data acquired through ESI MS/MS coupled with stable isotope labelling techniques from three studies was analysed using the smoothing spline mixed effect method (SME). This is a pilot study applying time course label enrichment of multiple surfactant PC species as biomarkers to phenotype animal models and patients with various lung diseases. There is no effective software for data processing mass spectrometry analysis of time series substrate incorporation into multiple molecular species products. Consequently, a bioinformatics platform Lipidome Labelling was developed to facilitate data analysis of dynamic labelling studiesand was used for the following biomarker discovery analysis. Time course methylD9 label enrichment of 16 PC species was modelled using SME method for all the subjects of group pairs in three different studies, which makes the foundation of the multivariate time course biomarker discovery analysis for lung diseases. In a study, a Ft statisticwas calculated as the scaled difference between the time course label enrichment of two contrasting groups for a single PC species, and the Ft statistics for all the species were ranked and according to their significance in distinguishing the two groups, for potential biomarker identification. The surfactant PC kinetic biomarker analysis of a mouse model shows that time course label enrichment of multiple PC species significantly differentiated between wild type mice and granulocyte macrophage colony stimulating factor (GM-CSF) beta chain knock out mice. However, this methodology failed to provide useful biomarkers in the clinical conditions examined, preterm infants at risk of neonatal respiratory distress syndrome and adult patients with acute respiratory distress syndrome compared with healthy volunteers. While differences could be determined between grouped data, subject heterogeneity precluded provision of diagnostic individual for individual patients.

Epithelial cell (EC) sheet engineering is currently of interest for both regenerative medicine and drug research. Existing cell models for asthma research lack the complexity of heterogeneous tissue, while animal models provide complexity at the expense of molecular pathway accuracy via genetic drift. New models that are both complex and accurate are needed to improve efficacy testing and increase drug translation to the clinic. Acoustofluidic (AF) levitation is the latest, non-invasive cell manipulation technique, that uses a piezoelectric transducer and resonator chamber to create an ultrasonic standing wave field (USWF) to trap cells within a pressure node. AF devices have previously been used to engineer 3D cartilage for transplant, create hepatocyte spheroids, and spatially organise cells within an extracellular matrix to create a vascular co-culture model. This project proposes novel microfluidic AF device designs for EC sheet engineering, utilising inexpensive glass capillaries as bioreactors, with potential for scalability by operating multiple devices in sequence. Levitated EC sheets could be used to engineer scaffold-free, autologous, multilayered, retinal and myocardial constructs for transplant, or more replicative in vitro research models. Bronchial epithelial cells (BECs) were levitated to continue work by Dr Tait developing a direct-contact airway mucosa co-culture model using similar AF devices. The final developed AF devices were able to levitate single BECs for one hour to create singular or multiple BEC sheets which satisfied morphological and functional requirements, such that they behave as cohesive sheet layers that move collectively via plithotaxis. The formation of BEC sheets within AF devices was visualised using immunofluorescence (IF); the surface area was then calculated using ImageJ software. Focal plane adjustments while levitating and IF adherens junction (AJ) staining post-seeding into collagen-coated wells confirmed that BEC sheets were sufficiently planar. Successful adhesion of BEC sheets to collagen before fixation was used as a blunt measure of viability. Transepithelial electrical resistance (TER) of long term BEC sheet cultures was measured using chopstick electrodes and voltohmmeter. TER readings after 11 - 13 days were indicative of tight junction and barrier formation. This study presents the first successful application of an inexpensive, glass capillary-based AF device for EC sheet creation, with potential for higher EC sheet output and easier sheet handling and transfer via a microfluidic system. Further work is needed to confirm the morphology and functionality of successfully levitated EC sheets, as well as experimentation using additional cell types to demonstrate the versatility of AF devices.

Respiratory virus infection is a common cause of hospitalisation in adults. New molecular platforms have been developed that can be deployed as point-of-care tests (POCT). Use of molecular POCTs for respiratory viruses may improve clinical care. This thesis explores the clinical impact of molecular point-of-care testing for respiratory viruses in adults hospitalised with acute respiratory illness. We did a large pragmatic, open-label, randomised controlled trial of routine molecular POCT for respiratory viruses, compared with routine clinical care, in adults presenting with acute respiratory illness to a large teaching hospital in Southampton over two winter seasons (the ResPOC trial). We recruited 720 patients of which 714 were included in the modified intention-to-treat analysis (360 assigned to POCT and 354 to routine care). The proportion of patients who received antibiotics was 84% in the POCT group compared with 83% in the control group (p=0.84). Of those who received antibiotics, 17% in the POCT group received a single dose or < 48 hours of antibiotics compared with 9% in the control group (p=0.0047). Mean length of stay was shorter in the POCT group (5.7 days) than in the control group (6.8 days; p=0.443). Appropriate antiviral treatment of influenza-positive patients was more common in the POCT group (91%) than in the control group (65%; p=0.0026). We went on to show that POCT with a turnaround time to results of < 1.6 hours was associated with higher rates of early hospital discharge and early discontinuation of antibiotics compared to longer turnaround times. To further explore attitudes to neuraminidase inhibitor use in influenza treatment, an online, cross-sectional questionnaire-based survey of self-reported prescribing practice using clinical scenarios was distributed to frontline clinicians. There were 237 respondents. Adherence to national treatment guidelines in the clinical scenarios ranged from 56% to 72% with considerable variability between specialities. Using ResPOC trial data, I also examined the reliability of pneumonia diagnoses on discharge documentation. 28% of patients with a diagnosis of pneumonia had no radiological evidence of pneumonia. 35% of patients with clinico-radiological evidence of pneumonia did not have a diagnosis of pneumonia recorded. In conclusion, while routine molecular POCT for respiratory viruses did not reduce the proportion of patients treated with antibiotics, POCT led to higher use of single doses or brief courses of antibiotics. POCT was also associated with a reduced length of stay and antiviral use and appeared to be safe. Rapid testing turnaround times were associated with better outcomes and unlikely to be achievable with centralised laboratory testing; this suggests that viral diagnostics should be performed at the point-of-care. The trial data also showed frequent misclassification of pneumonia diagnosis on discharge documentation and this may have clinical, financial and research data implications. A large survey of frontline hospital physicians suggested that guideline adherence in influenza treatment was sub-optimal but strategies that promote rapid diagnostic testing such as molecular POCT may improve adherence.

Chronic obstructive pulmonary disease (COPD) is a life-limiting illness characterised by progressive breathlessness and chronic cough that affects around 3 million people in the United Kingdom (UK). Patients with COPD have a high symptom burden, which is typically managed by means of aggressive and invasive treatments as patients approach the end of life. Although mortality rates are high, identifying with accuracy when patients are approaching the end of life is difficult and so there is uncertainty on when and how to initiate conversations about appropriate care plans with patients. To date, little research has focused on understanding patients’ and clinicians’ thoughts about the timing and nature of palliative care conversations. This study aimed to understand COPD patients’ preferences for palliative care discussions; and explore clinicians’ opinions and experiences when holding these conversations. The study is divided into 4 different phases that complement and inform each other. Phase 1 consisted of a systematic literature review that explored current evidence about palliative care discussions in COPD. The second phase looked at COPD patients’ preferences for palliative care discussions with clinicians. Thirty three patients at different stages of disease severity were interviewed, which included 8 patients with mild, 15 patients with moderate and 10 patients with severe and very severe COPD. Interviews used a semi-structured approach and data analysis was guided by interpretative phenomenological analysis (IPA). The third phase included interviews with 14 healthcare professionals that provided direct care to COPD patients. Clinicians from four different professional backgrounds were interviewed, including: general practitioners, practice nurses, COPD specialist nurses and COPD consultants. Interviews explored clinicians’ thoughts and experiences when holding conversations with patients, and were analysed using a thematic analysis approach. The fourth and last phase of the study comprised the data integration of the three research phases described above. The findings from the previous phases were combined using triangulation methods, integrated and discussed, to clearly answer the research question. The overall findings suggest that palliative care discussions were conducted by unfamiliar clinicians to the patient, at a late stage in the illness trajectory and when patients were admitted to hospital. Late discussions seemed to be caused by difficulties in timing and initiating discussions, service rationing and clinicians’ and patients’ poor understanding about palliative care and COPD. As an example, palliative care was seen as end of life care and exclusive of life-sustaining treatments. Poor understanding about palliative care and COPD seemed to increase clinicians’ and patients’ reluctance in discussing palliative care, undermining open discussions about preferences for future care and the development of action plans. Furthermore, data integration suggests that patients and clinicians had opposing perspectives about the optimal timing and nature of discussions, which increased the complexity of conversations and prevented their start. In contrast to current practice, some patients and most clinicians recommended early, gradual and informative discussions about palliative and future care/treatments. Early discussions were thought to facilitate the initiation and conduct of discussions and to reduce their emotional impact, enabling patients to participate fully. Thus, early discussions provide an opportunity for clinicians to understand how patients view and experience their condition and its treatments, and offer a treatment philosophy that suits patients’ preferences and is guided by principles of Burden of Treatment theory. The current approach when discussing palliative care advocates a sudden change in care, from aggressive treatments to end of life care and does not meet patients’ needs. A new way of thinking about consultations where there is a dialogue that focuses on addressing patients’ needs, symptoms and priorities, whilst reducing treatment burden throughout the disease course is recommended. This new approach can create individualised care plans that meets patients’ needs and preferences.

Chronic rhinosinusitis (CRS) is a chronic inflammatory condition affecting the lining of the nose and paranasal sinuses. It is the second most common chronic disease worldwide, and impacts significantly on patients' quality of life and healthcare resources. CRS is subdivided into two main disease categories: CRS with and without nasal polyps (CRSwNP and CRSsNP respectively). It is well established that bacteria, most notably Staphylococcus aureus (S. aureus), play an important role in the pathogenesis of CRS. Whilst much emphasis has been placed on surface bacteria, intracellular bacteria are gaining more prominence in relation to resistant disease. The intracellular environment may provide a protective niche for pathogenic bacteria to evade host immunity, and provide a reservoir for re-infection. Recently published findings from the Southampton Upper Airway Research Group reported the novel finding of intracellular S. aureus within nasal polyp mast cells, in addition to epithelial cells. Furthermore, the presence/addition of Staphylococcus aureus Enterotoxin B (SEB) further promoted the internalisation of S. aureus into mast cells. The aim of this work was to further characterise the host response towards S. aureus and its toxins, with particular emphasis on S. aureusmast cell interactions. A prospective study was performed using ex-vivo sinonasal mucosa and nasal polyp tissue from CRS patients, and sinonasal mucosa from non-CRS patients undergoing trans-sphenoidal pituitary surgery as a control. Pro-inflammatory cytokine profiles of these tissue sub-sites were measured using real time quantitative polymerase chain reaction (RT-qPCR). An explant tissue model was developed to study the immune response of nasal polyps, and control samples to SEB, with the resultant host immune response measured using RT-qPCR and Luminex. An in vitro cell culture model was developed to characterise interactions between a CRS-specific S. aureus isolate and the RPMI-2650 epithelial cell line, HMC-1 mast cell line and LAD2 mast cell line. S. aureus was cultured to the mid-log phase and combined with all three cell lines, at set time-points and multiplicity of infection ratios. Intracellular uptake of S. aureus was examined using confocal laser scanning microscopy, intracellular viability and its release was examined using colony forming unit (CFU) enumerations, and the associated host immune response was measured using RT-qPCR and Luminex. Study of the potential phenotypic change to S. aureus, from its continual uptake and release from mast cells, was undertaken in both HMC-1 and LAD2 cell lines, utilising CFUs, RT-qPCR and Luminex. For the study of IgE sensitisation and its effect on the activation of S. aureus infected LAD2 cells, the following techniques were used; LDH assays, degranulation (β-hexosaminidase) assays, protein kinase phosphorylation, RT-qPCR and Luminex. In comparison to control patients, CRSwNP patients display upregulated pro-inflammatory cytokines (IL-5, IL-8) toll-like receptors (TLR-4) and matrix metalloproteinases (MMP-28). In many cases the nonpolypoidal sinonasal mucosa of CRSwNP and nasal polyps display strikingly similar immune profiles, with no statistical difference found between the two sites. SEB was found to significantly upregulate nasal polyp gene expression ratios of IL-5, IL-17A, TNFα, TGF-β, and supernatant protein concentrations of IFNγ, IL-5, IL-17A, and TNFα. In cell line culture experiments, RPMI-2650 epithelial cells, as well as HMC-1 and LAD2 mast cells, readily internalised S. aureus. This intracellular uptake was significantly enhanced in the presence of SEB for both epithelial and mast cells at 24 hours. Intracellular S. aureus was found to be viable and capable of release, rapidly replenishing previously eradicated extracellular bacterial populations. Upon S. aureus exposure, both mast cells (HMC-1) and epithelial cells (RPMI-2650) contributed to inflammation through pro-inflammatory cytokine release vi into the culture supernatant (IFNγ, TNFα, IL-17A, IL-1β and IL-6). S. aureus was able to significantly downregulate both the gene expression (IL-8, IL-1β, TNFα, TGF-β1 and IL-5) and protein release (TNFα) of LAD2 mast cells through the sequential repeated intracellular uptake and release of S. aureus. In the presence of prior IgE sensitisation, S. aureus infected LAD2 mast cells were able to limit degranulation, the host immune response (TNFα gene expression and protein release), and the phosphorylation of Atk2 and GSK-3α/β. These findings demonstrate the importance of S. aureus and its toxins in the development of a chronic inflammatory reaction in CRS patients. Epithelial and mast cells appear to provide a protective niche, shielding S. aureusfrom immune mediated clearance, as well as acting as a reservoir of infection that can replenish eradicated bacterial populations. Through prior IgE sensitisation of mast cells, S. aureus was able to manipulate the subsequent host immune response, favouring its own survival. A similar response was also seen following repeated uptake and release of S. aureus from mast cells, suggesting bacterial phenotypic change. This is of particular relevance in patients with elevated IgE levels, as is seen in patients with S. aureus colonisation where IgE forms against both S. aureus and its enterotoxins. This may well facilitate the ongoing survival and submucosal persistence of S. aureus, and could contribute towards the development of a chronic disease process. These patients are likely to benefit from aggressive medical therapy, particularly in the post-operative period, to eradicate S. aureus, in order to improve long-term treatment outcomes.

Wheeze and asthma are major health problems worldwide, affecting all age groups. Severe asthma and asthma exacerbations represent particular problems because they are associated with high morbidity and healthcare costs. This thesis used data collected as part of the EuroPrevall and UBIOPRED studies to provide new insights into wheeze and asthma across the life course. Areas explored included risk factors for preschool wheeze, the relationship between atopy and disease severity and risk factors for exacerbations in patients with severe asthma/preschool wheeze. The EuroPrevall birth cohort consisted of 12,049 infants from nine European countries. Data on wheeze in the second year of life was available in 8775 (72.8%). The prevalence of wheeze varied considerably across Europe, ranging from 1.7% in Lodz (Poland) to 17.2% in Reykjavik (Iceland). Risk factors for wheeze in the second year of life included lower respiratory tract infections, postnatal maternal smoking, day care attendance and male gender. However, their importance varied between centres suggesting that unique risk factors operate in different countries. In the UBIOPRED study, participants with mild to moderate and severe asthma/preschool wheeze were recruited into adult, school and preschool age cohorts. At baseline, a detailed asthma and allergic disease history was taken. Skin prick testing, specific IgE measurement and component resolved allergen diagnostics (ISAC Chip®) were performed. The severe cohorts were followed up after 12-18 months. Clinical clusters and allergic sensitisation clusters were generated. The prevalences of allergic disease and allergic sensitisation did not differ significantly according to asthma/wheeze severity in any age group. A history of previous exacerbations and poor asthma control were risk factors for future exacerbations across the life course. Rates of prospective exacerbations did not differ between clinical or allergic sensitisation clusters. Further research is needed to determine whether novel biomarkers can more accurately predict asthma outcomes than clinical parameters.

Carriage of Neisseria lactamica occurs naturally at high frequency in infants and low frequency in young adults. There is an inverse epidemiological relationship between N. lactamica carriage and disease caused by Neisseria meningitidis (meningococcus). Serogroup B meningococci remain the dominant cause of invasive meningococcal disease in the developed world and have frustrated the production of polysaccharide-conjugate vaccines. While two, recombinant, OMV based vaccines (Richmond et al., 2012; Vernikos and Medini, 2014) have been created and elicit immunological responses, they are less effective on infants (one of the groups most at risk of IMD) and have limited effect on meningococcal carriage and subsequently, on herd immunity. A human experimental challenge study in which healthy, young adult volunteers were inoculated with N. lactamica Y92-1009 showed that carriage of N. lactamica both displaced and inhibited reacquisition of wild type N. meningitidis, and although rare, co-colonization of the two species was also observed in a small number of cases (Deasy et al., 2015). This study provided the opportunity to investigate whether there is a genomic basis for N. lactamica's effect on meningococcal carriage as the mechanism for this interaction remains unknown. Secondly, the use of whole genome sequencing, paired with mutation analysis via the breseq pipeline (Barrick et al., 2014) will comment on the mutability of N. lactamica, a potential bacterial medicine, during 6 months of in vivo, human challenge. Thirdly, this allows us to track the within-host microevolution of an identically administered commensal Neisseria spp. over the course of 6 months of carriage (chapter 5). Isolates obtained from individuals who were co-colonised by N. meningitidis and N. lactamica for a prolonged period were examined for evidence of the effect of recombination (r/m) as well as loci affected by it (chapter 6). In addition to the majority of volunteers who solo carried N. lactamica Y92-1009. Recombination was determined for; volunteers in which inoculated N. lactamica was the sole Neisseria spp. detected, seven, artificially inoculated, N. lactamica/meningococcal co-carriers and two extra volunteers who were naturally co-colonised. Using ClonalFrameML (Didelot and Wilson, 2015), we detected minimal homologous recombination events among N. lactamica Y92-1009 and no examples of interspecific allele transferred with co-colonising meningococci. In contrast, we found evidence of a dynamic, interspecific relationship and a number of recombination events occurring among co-colonised volunteers with naturally acquired Neisseria. A separate, short term clinical trial utilizing multiple colony sampling (chapter 4) examined the difference in mutational profiles of longitudinal samples N. lactamica strain Y92-1009 sourced from in vitro conditions versus in vivo conditions over one month. Larger numbers of SNPs, nonsense and recurring mutations were observed among the in vitro cohort and the quantity/diversity of phase variable mutations was more pronounced among the in vivo cohort. Chapters 4 and 5 are supported by a highly-accurate reference genome. The sequencing, assembly, annotation and characterisation of the first complete N. lactamica Y92-1009 genome is described in chapter 3 (Pandey et al., 2017). This chapter also revealed the presence of a large but uncharacterised prophage sequence in the strain. The very first example of a species encompassing, pan genomic analysis of N. lactamica (chapter 7) revealed that N. lactamica Y92-1009 possess fewer unique genes/alleles than other members of the species with no virulence factors detected among the results. In conclusion, the N. lactamica Y92-1009 genome is a self-curated system with plastic elements that (like other Neisseria spp.) could facilitate rapid changes in expression via its phase variable elements. However, it appears to have remained genetically stable during the 6-month course of carriage in human volunteers. Demonstrating little recombination, no interspecific gene transfer with co-colonising meningococci and an average mutation rate for a Neisseria species. While efforts need to be made to improve the acquisition and retention of carriage, N. lactamica appears to be a safe, naturally competent, potential bacterial therapeutic, capable of a broadspectrum reduction of meningococcal carriage.

Influenza infection is a major contributor to global mortality and morbidity, with high potential for pandemic and vaccine evasion. Natural Killer (NK) cells are innate anti-viral effector cells but their functions in respiratory influenza infection are poorly understood. NK cells have been implicated in the control of influenza in mouse models but their regulation in the human respiratory environment is still an area of study. For instance, phenotypically and functionally unique resident NK cell populations have been described in the liver and uterus, but have not yet been explored in the human lung. The aim of this thesis was to investigate the human lung NK cell phenotype and function during respiratory infection and disease, primarily focusing on influenza infection. To do this, NK cells were characterized from macroscopically normal human lung tissue by multi-parameter cytometry and NK cell function characterized in an ex vivo tissue explant model of influenza infection. NK cell function was further characterized by autologous co-culture of peripheral NK cells with influenzainfected monocyte-derived macrophages (MDMs) and primary bronchial epithelial cells (PBECS). Cytotoxic and cytokine responses of NK cells were measured by a combination of flow cytometry and enzyme-linked immunosorbent assay (ELISA). Mechanisms of NK cell activation to IAV-infected macrophages were further explored including activating and inhibitory contact with infected cells and cytokine release. Both lung and peripheral NK cells were found to mediate strong anti-viral responses to influenza infection; with increased IFN-γ, Gzm-B and measures of degranulation. Furthermore, mechanistic studies demonstrated that physical contact between NK cells and influenza-infected MDMs was essential to NK cell activation, despite a strongly pro-inflammatory environment. However, NKp46 and NKG2D ligands were redundant in stimulating NK cell activation towards infected MDMs, demonstrating complicated and extensive regulation of NK cells activation by infected macrophages. The majority of human lung NK share a phenotype with circulating NK cells, however, this thesis identified novel lung resident NK cell populations. Lung resident (CD49a+) NK cells were found to respond more potently to influenza infection than non-resident (CD49a-) NK cells. This suggests that resident, trained NK cell populations are present in the human lung and may provide early and important control of viral infection. CD49a+ lung NK cells were also increased in donors with more severe COPD, indicating possible modulation of lung resident NK cells in chronic disease. Overall this thesis presents evidence that NK cells play an important role during human influenza infection through both cytotoxicity and IFN-γ production in the human lung. This work has also highlighted the differences between lung resident and non-resident NK cells in terms of phenotype and function, which may have important implications for understanding mucosal immunity in the lung.

Exacerbations landmark the natural course of COPD and are associated with worsening airway obstruction, impact patients health status and have a considerable socio-economic impact. There is a gap in our understanding of different phenotypes of COPD exacerbations and therefore the strategies for treatment of exacerbations have remained unaltered for many years. Diagnosing an exacerbation event is largely subjective and depends on the assessment of presenting symptoms. Treatment of an exacerbation event is often generic and does not take into account different inflammatory mechanisms and underlying aetiological factors. The aim of this work was to determine whether clinically available airway and blood biomarkers can be used, separately or jointly, to identify exacerbations of COPD and in particular bacterial infection at exacerbations. The analyses were conducted on data collected from the Acute Exacerbation and Respiratory InfectionS in COPD cohort (AERIS) - a longitudinal observational study conducted from 2011-2014. The cohort consisted of patients with moderate to very severe COPD with a previous history of at least one moderate exacerbation. Patients were seen during clinical stability on a monthly basis and at exacerbations. Exacerbation events were monitored prospectively and patients were seen within 72 hours of an onset of the exacerbation event. Pulmonary function was assessed, and sputum samples were collected on a monthly basis and at exacerbations. Sputum samples were processed for microbiology and biomarkers. Blood samples were collected quarterly and at exacerbations and processed for biomarkers. The AERIS study was delivered at the standard of a randomised controlled trial with the rigorous monitoring and data cleaning, therefore, the data quality and the study conclusion are sufficiently robust to set up the vaccine study. This work for the first time describes an association between the persistence of eosinophilic inflammation in COPD during clinical stability with an increased likelihood of eosinophilic exacerbations, and an association between eosinophilic inflammation, seasonality and bacterial presence. Namely, bacterial infection at exacerbation was higher in the Winter than Summer in patients with predominantly blood eosinophils≥2% at stable visits. A larger proportion of the exacerbations with raised blood eosinophil ≥2% were observed during the Summer compared to Winter season. The findings confirm that sputum purulence can be examined by the five-graded Southampton sputum colour chart and the sputum colour grade was associated with airway neutrophils and airway bacterial presence at exacerbations. The numeric change in sputum colour was associated with airway bacterial presence; the higher the numeric change the greater was the frequency of airway bacterial detection. Higher sputum colour, higher CRP, higher fibrinogen, higher blood neutrophils and lower blood eosinophils were useful in identifying an exacerbation event with airway bacteria. Therefore, these observations support the view that there are markers that are readily accessible in routine clinical care. These biomarkers reflect airway and systemic inflammation, they can be used in the diagnosis of airway bacterial infection at exacerbations and so may aid the management of COPD exacerbations. Further work to confirm the impact of these findings in improving clinical outcomes in intervention studies is required.

A disintegrin and metalloproteinase 33 (ADAM33) was identified in 2002 as an asthma susceptibility gene; it has also been shown to be associated with bronchial hyperresponsiveness (BHR), atopy and lung function decline. Studies in asthmatic patients have identified a soluble, enzymatically active form of the protein (sADAM33) which is increased in asthmatic bronchoalveolar lavage fluid, and correlates with disease severity. In support of a functional role for ADAM33 in asthma, transgenic mice expressing human sADAM33 exhibit increased airway remodelling and susceptibility to allergic airways disease, whereas mice lacking endogenous Adam33 are relatively protected against allergic airways disease. The aim of this investigation was to use unbiased approaches, specifically, next generation RNA sequencing to investigate airway responses involving ADAM33. The first objective was to investigate the transcriptome of transgenic mice where sADAM33 expression had been induced under allergen naive conditions to identify mechanisms by which this may promote susceptibility to allergic airways disease. The second objective was to study how baseline and allergic airway responses were altered in response to Adam33 abrogation, investigating the transcriptome of Adam33 knockout and wildtype mice that had been challenged with saline or house dust mite to identify ADAM33 dependent allergic mechanisms. The first investigation identified a novel association between expression of human sADAM33 in murine airways and the induction of an immune response-associated gene signature, including cytotoxic lymphocyte markers, and an interferon inducible gene signature. Further exploration of this signal identified a significant increase in granzyme B mRNA and protein in the lungs of sADAM33 mice, which was accompanied by an altered natural killer cell phenotype. Natural killer cells in sADAM33 mice retained their expression of the activation marker NKp46 and homeostatic marker KLRG1, identifying an increased NKp46+ KLRG1+ population, which preferentially expressed granzyme B. These results suggest that sADAM33 in the airway, may drive natural killer cells towards a more mature cytotoxic phenotype, which could be influential in subsequent allergic responses, identifying a role for sADAM33 in disease promotion. RNA sequencing of Adam33 knockout and wildtype mice identified only 6 genes (inclusive of Adam33) differentially expressed between saline challenged mice, suggesting a requirement for an environmental stimulus to drive differential responses in these models. House dust mite challenge drove a clear separation in the based upon genotype at the whole transcriptome level, identifying a substantial differential response to allergic sensitisation between Adam33 knockout and wildtype mice. Pathway analysis identified an ADAM33-dependent metabolic response, with a particular emphasis on oxidative phosphorylation complexes, implicating mitochondrial mediated mechanisms of disease. Further work is required to dissect these mechanisms, identifying the cellular source and their involvement in allergic airways disease. The similarity between the allergen naive wildtype and Adam33 knockout mice, and the identified role for ADAM33 in disease predisposition and allergic responses, suggest that ADAM33 could be a suitable target for future asthma modifying therapies.

Chronic rhinosinusitis (CRS) with or without nasal polyps (NP) affects up to 15% of the UK population significantly affecting quality of life and impacting heavily on health care resources. Despite millions of pounds spent on research each year, the underlying cause of CRS remains unknown. Bacteria have been implicated in playing a role in mediating the ongoing inflammation in CRS. Bacteria possess the ability to quickly adapt to environmental changes, readily interchanging between planktonic, biofilm and intracellular profiles. Emerging evidence suggests that CRS is the result of a locally dysregulated innate immune system caused by changes in bacterial profiles in a bid for survival. The work presented in this thesis aimed to clarify this proposed theory through characterisation of bacterial profiles in CRS and analysis of the effect on the local innate immune system at a cellular level. A prospective study was performed using ex-vivo sinonasal mucosal and NP tissue from CRS patients, and sinonasal mucosa from non-CRS patients undergoing transsphenoidal pituitary surgery as a control. Tissue was analysed using the LIVE/DEAD® BacLight™ viability kit, fluorescent in situ hybridisation, scanning electron microscopy, transmission electron microscopy, confocal laser scanning microscopy and immunohistochemistry. An explant tissue model was developed to explore histological changes at the host-environment interface after addition of exogenous Staphylococcus aureus (S aureus) and Staphylococcus enterotoxin B (SEB). An in vitro cell culture model was developed to investigate cellular changes resulting from mast cell-S aureus interactions. Surface-related biofilms were identified on CRS sinonasal mucosa. S aureus was the commonest microbe identified. Sodium nitroprusside appeared to have little or no effect on dispersal of surface-related CRS biofilms. In NP, bacteria were sub-epithelial and within mast cells (intracellular). This is the first reported study to observe intracellular S aureus within mast cells in NP. The presence of SEB significantly increased the internalisation of S aureus into mast cells. S aureus entered mast cells through phagocytosis and via extracellular traps. Proliferating intracellular S aureus led to mast cell expansion and eventual rupture, seeding viable S aureus and proinflammatory mediators into the extracellular space. These findings demonstrate the ability of S aureus in CRS to manipulate the host's innate immune system in order to facilitate survival. Adaptation of bacteria into different profiles results in different pathophysiological effects. The intermittent dispersal of active planktonic bacteria from surface-related bacterial biofilms on nonpolypoidal mucosa mediates the ongoing chronic inflammation and symptom relapse in CRS. Intracellular S aureus within mast cells could act as a reservoir for bacteria constantly seeding into the extracellular environment leading to the build-up of proinflammatory cytokines and mediators, thus promoting tissue oedema and potentially nasal polyp growth. These findings suggest that NP formation may be an indirect consequence of a S aureus survival strategy to evade host defences.

Streptococcus pneumoniae is both a common inhabitant of the human nasopharynx and a leading cause of morbidity and mortality. Characterisation of pneumococcal populations derived from asymptomatic carriage and invasive disease during the introduction of a 13-valent pneumococcal conjugate vaccine (PCV13) allows the impact of the vaccine to be assessed and may provide information with which to inform public health policies. Streptococcus pneumoniae isolated from the nasopharynx of healthy children were collected alongside isolates found in invasive pneumococcal disease (IPD) from the same geographical location over a ten year period. Carriage rates and IPD incidence rates were examined to assess the impact of PCV13 on both populations. Changes to the serotype distribution in both the carriage and the disease populations were compared and a molecular investigation was performed to ascertain similarities between the populations. Pneumococcal carriage and IPD incidence caused by PCV13 serotypes decreased following PCV13 introduction however an increase in carriage and IPD incidence caused by non-vaccine serotypes was observed. Evaluation of serotype distribution revealed that three non-vaccine serotypes had significantly higher odds of featuring in IPD; 12F, 8 and 9N. Genotypic analyses showed that while 83% of IPD isolates had the same clonal type as carriage isolates, some molecular variations within prevalent carriage isolates were not seen in the IPD population. The increase in carriage and IPD caused by non-vaccine serotypes is compromising the benefits of PCV use. Continued surveillance is required to safeguard public health.

Mast cells (MCs) are classically involved in the pathogenesis of allergic asthma, however MCs also have a key role in innate immunity with an emerging role in viral immunity. During allergic asthma MCs localise in greater numbers to the bronchial epithelium which is the principal site of human rhinovirus (HRV) infection. HRVs are a major viral trigger of asthma exacerbations via mechanisms that are not completely understood. MCs are susceptible to HRV infection but their role in antiHRV responses is unknown. HRV infection of the bronchial epithelium triggers the release of the epithelial derived cytokine IL-33, which induces Th2 cytokine release from target cells with MCs being the major target of IL-33 in allergic asthma. I hypothesised that HRV infection induces MC anti-viral responses and also modulates IL-33-dependent Th2 responses in MCs. The LAD2 human MC line and/or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV (control infection) with or without IFNβ, IFN-γ, IFN-λ or IL-33. Twenty-four hours following HRV infection, anti-viral and Th2 immune responses were assessed by RT-qPCR, MSD, ELISA and flow cytometry. Viral replication and release were determined by RT-qPCR and TCID50 assay respectively. HRV infection induced the expression of IFN-β and IFN-λ and the induction of IFN stimulated genes (ISGs). Despite this MCs were permissive for HRV replication and the release of infectious HRV particles. This was confirmed in CBMCs. To determine the contribution of endogenous anti-viral responses, CBMCs were treated with a type I IFN receptor blocking antibody. Treatment with the blocking antibody failed to significantly increase HRV replication and release suggesting endogenous type I IFN responses were insufficient to protect MCs against HRV infection. Therefore, in order to enhance anti-viral responses, MCs were treated with exogenous IFN-β, IFNγ or IFN-λ. The induction of ISGs was enhanced by IFN-β and IFN-γ but not by IFN-λ. In addition, IFN-β treatment significantly suppressed viral replication and the release of infectious virus particles. To investigate the impact of HRV on IL-33-mediated Th2 responses, MCs were treated with IL-33 during HRV infection. IL-33 treatment induced a concentration-dependent increase in the release of IL-5 and IL13 but this was not modulated by HRV. However, IL-33 treatment increased ICAM1 expression, a receptor for HRV entry, and enhanced HRV-mediated induction of IFNβ and ISGs. This resulted in a significant increase in HRV replication but prevented significant release of infectious HRV particles. These findings show for the first time that MCs mount anti-viral responses to HRV infection and that HRV-induced IFN-β production is enhanced by IL-33 treatment. In severe asthma, which is associated with impaired bronchial epithelial IFN responses and an increase in the localisation of MCs to the bronchial epithelium, MCs may aggravate HRV-induced exacerbations. However, IL-33 released from the epithelium may protect MCs against productive HRV infection. These findings may have important implications in HRV-induced asthmas exacerbations and the impact of novel asthma therapies particularly anti-IL-33.

Our knowledge and understanding of asthma have evolved over time, leading to new and improved treatments for this disease. Despite existing treatments however, there remains to date a significant proportion of asthmatics who remain poorly controlled, with unmet needs. Most existing treatments are based on the Th2-driven inflammation model of asthma, however there is increasing recognition of the importance of the epithelium in asthma pathogenesis. It has been proposed that the asthmatic epithelium is chronically damaged and unable to repair, with increased permeability as a result. Existing treatments do not address the epithelial damage directly, however there are now available recombinant growth factors that have been shown to have beneficial effects on epithelial healing. Our hypothesis was that modification of the epithelium, in effect boosting its repair using recombinant human keratinocyte growth factor (rhKGF), would lead to improvement in clinical parameters. This was explored in several fashions. Firstly a randomised, double-blind, placebo-controlled clinical trial was performed using 20 poorly controlled, moderate asthmatics, with the active treatment group receiving parenteral rhKGF. Assessments before and after drug administration included objective, clinically relevant, measures of asthma such as airway hyperresponsiveness (AHR) measurements, spirometric measures, exhaled nitric oxide measurements and peak flow recording. Subjective, patient-centred assessments were also made using questionnaires to assess asthma control and quality of life, and bronchoscopy was performed to obtain samples to measure biological effects of the drug. KGF treatment resulted in a significantly greater improvement in the primary outcome of mannitol AHR, together with greater improvements in quality of life in the active treatment group compared to placebo. Other features (such as methacholine AHR, asthma control questionnaire scores, spirometric values, exhaled nitric oxide and peak flow variability) did not differ significantly between the groups, although this may be due to a greater than expected placebo response. Biological outcomes also did not differ significantly between the groups, although this may have been due to the sampling time-point used. Concurrently to the clinical trial above, in vitro experiments were performed on cell cultures of epithelial cells from asthmatic and healthy donors, to verify and further explore the effects of KGF on an asthmatic epithelium. Specifically mechanical wounds were inflicted on the cultures, with assessment of the repair process using wound imaging, measurement of trans-epithelial electrical resistance (TER) and permeability to FITC-labelled dextran, in the presence and absence of KGF. As a subset of these experiments, some cultures were exposed to mechanical compression using air pressure, as a mimic for bronchoconstriction, to see if KGF was effective in these circumstances. Results confirm a biological effect for KGF on wound repair in the asthmatic epithelium, which can also partially overcome the deleterious effect of compression on wound healing. An intrinsic difference in wound healing between asthmatic and healthy cohorts, as previously reported, was not apparent. Lastly the potential of nuclear medicine imaging, to assess epithelial permeability, was explored, for its potential use in future studies of asthma treatments addressing the epithelium directly. Unfortunately this was halted after a pilot study suggested potential methodological flaws – the results and conclusions from this pilot study are presented here, with suggestions for future studies in this area.

The initial part of this thesis comprises of a thematic synthesis of qualitative research followed by a qualitative study. The thematic synthesis aimed to gain an understanding of how participants with long term health conditions and physical health difficulties, experience mindfulness interventions. Thomas and Harden’s (2008) three step thematic synthesis method was utilised, in order to synthesise the qualitative findings of 20 qualitative studies. This process revealed five key themes: 1) Perceived barriers to participation and practice: 2) Positive consequences and benefits of participation: 3) Group attributes: 4) Negative experience and 5) Home practice and application to life. Mindfulness based interventions are acceptable for individuals with long term and physical health conditions. Individuals can experience both positive and negative experiences of mindfulness interventions. The group aspects of these interventions are valued by participants for their support and social aspect; however some patients can experience a negative effect, which requires further exploration. The needs of patients with respiratory conditions and dual mental health difficulties are not adequately catered for. The literature investigating mindfulness and respiratory conditions, especially asthma, is limited. There is only one study to date, which has looked at the effectiveness of mindfulness compared to treatment as usual in a randomised control trial for asthma, which has shown promising results. The present study aimed to explore poorly controlled asthma patients’ experience of a brief mindfulness group. A qualitative approach was taken to elicit the views of adults with poorly controlled asthma from a difficult asthma clinic, who attended a four week mindfulness course. A focus group and nine semi structured interviews were conducted and analysed using thematic analysis. The study found four overarching themes including: Wellbeing, Challenges, Acceptability and Integration. These results imply that despite the challenges, a mindfulness based intervention is acceptable for poorly controlled asthmatics. Furthermore, it had a positive impact on wellbeing and patients were able to integrate skills alongside existing treatment. Further exploration of tailored mindfulness based interventions for asthmatics would be beneficial. In particular, exploring both subjective experiences and impact on health related outcomes, as well as psychological barriers to attendance and practice.

Chronic obstructive pulmonary disease (COPD) is a collective term for a condition associated with irreversible airways obstruction causing breathlessness and risk of recurring chest infections, termed exacerbations. Malnutrition has been shown to play an important role in many chronic diseases, but in COPD the extent that differences in nutritional wellbeing determine the patient's risk of exacerbation, resilience to infection or response to treatment, has not been adequately addressed and so nutritional aspects of care have not been considered primary concerns in the management of the disease. COPD exacerbations are assumed a major driver for the progression of COPD and of healthcare utilisation. Current respiratory variables measured in routine clinical practice are insufficient to identify patients at risk of exacerbation in the short- to medium-term and hence interventions to ameliorate exacerbation risk are limited. There is a real need to explore other non-respiratory markers for risk assessment in COPD patients. Whilst nutritional assessment has been established as an important tool in understanding risk of long-term outcomes, especially mortality, its role in assessing patient's risk of and response to exacerbations is less well understood. This thesis sought to explore the nutritional status in COPD patients with different disease presentations and clinical outcomes, in order to identify nutritional markers of exacerbation risk. Analysis focused on exploring the relative importance of disease history on nutritional status at baseline (how does previous disease impact on nutritional status), associations between disease status and nutritional status in stable COPD and relationship between nutritional status and clinical outcomes like time to first exacerbation (TTFE) and exacerbation rate over 12 month. Systematic review of the literature demonstrated variability in methods used to assess structural and functional markers of lean mass of COPD patients, which limited the conclusions that could be made. To address this a comparison of methods used in COPD patients was devised and conducted, showing comparability of the results of these methods. To explore the relationship between the nutritional status and clinical outcomes in COPD, a longitudinal, 12 months prospective cohort study, comprised of 127 COPD patients with a previous history of exacerbations was carried out. Nutritional status was assessed using markers of body composition, appetite and functional capacity (grip strength, grip endurance, walk test) at baseline and quarterly basis. These nutritional markers were used to explore their relationship with past medical history, baseline disease markers (spirometry, inflammation) and clinical outcomes, with further assessment of their predictive value for clinical outcomes measured as TTFE.In the studied cohort, 7 to 25% men and approximately 33% women had lean depletion (depending on applied criteria), and unexpectedly, low lean mass was not associated with a shorter time to first exacerbation. Those who had poor appetite were at higher risk of exacerbation in the following month (HR 1.6, p=0.023), which was independent of the exacerbations history and disease severity. Functional limitations including stopping during the 6- minute walk test (HR 1.90, p=0.001) and distance of less than 350m (HR 1.95, p=0.002) were related to TTFE. As none of the domains was sufficient to independently identify patients at high risk of exacerbation with sufficient accuracy in the next month, a multicomponent approach was taken by combining appetite score, body composition and walk test. Poor nutritional status marked by two or more components showed a higher risk of exacerbation, compared with those identified by any single component (HR 3.47 p < 0.001). Use of history of exacerbation as additional component did not improve relevance to clinical outcomes and appetite score appeared to be of the greatest relevance to time to first exacerbation. In summary, the findings of this thesis demonstrate that nutritional status is not determined by the history of exacerbations, yet relates to markers of disease status in stable COPD and clinical outcomes in the future. Multicomponent nutritional assessment showed potential to identify patients with high risk of exacerbation in the near future, which was not possible when using standard respiratory measures alone. This thesis emphasises the role of assessing nutritional status in COPD patients and the relevance and need for recognizing different nutritional phenotypes in the disease management.

COPD is a heterogeneous disease with multiple clinical phenotypes and biological endotypes and the association between these are poorly understood. Novel quantitative analysis techniques permit objective measurements of pulmonary and extra-pulmonary manifestations of the disease. This thesis examines the role of quantitative CT analysis in exploring COPD, with the aim of showing that these techniques can potentially give important insights into the disease and explore the heterogeneity and underlying biology of the condition. CT image analysis was performed on two different cohorts of COPD subjects, which included quantification of emphysema, small airways disease and bronchial wall dimensions. Furthermore, novel analysis was also performed to quantify emphysema sub-types and body composition parameters. One of the cohorts was a small cross-sectional study of mild-moderate COPD subjects involving CT imaging and bronchoscopic sampling of the airways. BAL samples were analysed for inflammatory cytokines, white cell differential, matrix-metalloproteinases (MMPs) and microbiology. The other cohort was a larger 2 year longitudinal study of moderate-very severe COPD subjects who had CT imaging at enrolment and at 2 years. They also had careful characterisation with pulmonary function testing, exercise testing, sputum and blood analysis and clinical follow-up. The findings confirmed that CT image analysis could successfully measure emphysema and small airways disease with both having significant relationships with pulmonary function. Using these measures, I demonstrated a novel relationship between multiple MMPs and CT measured small airways disease, suggesting these proteases play an important role in the small airway remodelling that occurs in COPD. There were also associations between emphysema and MMPs, providing further evidence for their role in parenchymal destruction. In the larger cohort emphysema was independently associated with desaturation on exertion, but neither emphysema nor small airways disease had significant associations with blood or sputum inflammatory markers, sputum bacterial detection or exacerbation rate. On longitudinal analysis, emphysema progressed over a two period, although the causes for this were not apparent in my study. Quantification of airway dimensions were variable and unreliable and did not provide significant information about disease in either of my two cohorts. The findings from this study suggest that CT image analysis can be used to explore the disease and provides further rationale for future investigation into the use of image analysis in assessing different features of COPD.

Chronic obstructive pulmonary disease (COPD) accounted for 5.71% of all global mortality in 2015. Non-typeable Haemophilus influenzae (NTHi) is a genetically diverse opportunistic pathogen that colonises the respiratory tract and is linked to acute exacerbations in COPD which eventually lead to a fatal irreversible degradation of lung function. H. haemolyticus is the closest phylogenetic relative to NTHi and is also found to colonise the respiratory tract of subjects with COPD. However, H. haemolyticus is not thought to cause either invasive disease or exacerbations in COPD. The motivation for the work in this thesis is that there are currently no molecular or culture assays that can unequivocally differentiate these two species. The genetic diversity of NTHi has been reported. The objective of this study was to determine whether the genetic variation in atypical strains of the two species was sufficiently large to result in a genetic continuum that makes their delineation impossible and therefore questioning the current taxonomic classification. Whole genome sequencing data generated from 1460 isolates phenotypically identified as Haemophilus spp. were used to determine the genomic heterogeneity of NTHi and H. haemolyticus. This included assessing the utility of gene markers for example, fucK, smpB, iga, sodC, lgtC, hpd, omp2 and omp6, which have previously been shown to enable differentiation, as well as more extensive comparative genomic analyses. These data revealed the presence of previously poorly recognised atypical strains of NTHi and H. haemolyticus. Atypical isolates were initially characterised as those with an unexpected genotype for NTHi or H. haemolyticus as defined by previously described markers. The use of further adhesin genes and comparative genomics supported these findings. Comparison of NTHi and H. haemolyticus demonstrated distinctive clustering based on ANI with <92% similarity; a value below current taxonomic thresholds for species definition. In conclusion, despite extensive genomic variation in strains of NTHi they may be routinely separated from H. haemolyticus using whole genome analysis and multiple gene markers.

Anxiety is a key component of respiratory disorders, which can exacerbate symptoms as well as impact upon treatment outcomes. This PhD offers an original contribution to knowledge by demonstrating the clinical effects of acupuncture on anxiety related to two chronic respiratory disorders, (hyperventilation syndrome (HVS) and chronic obstructive pulmonary disease (COPD)). This thesis contains findings from two novel trials that have examined the use of body acupuncture and ear acupuncture for the treatment of anxiety associated with respiratory disorders. These studies are linked through one research question: does acupuncture, offered as an adjunctive treatment to physiotherapy, reduce anxiety in patients with common chronic respiratory disorders? Previous research examining acupuncture for the treatment of anxiety has focused on the treatment of generalised anxiety disorder, or anxiety related to exposure to anxiety provoking situations. The research included within this thesis has examined the use of acupuncture in a population of individuals with respiratory disorders to assess the feasibility of using acupuncture for anxiety in this population, and to enhance our understanding of its efficacy and clinical benefits. No previous respiratory acupuncture studies have been identified that were designed with anxiety as a primary outcome. Study 1 is a three-arm single-blind randomised controlled trial (RCT) of acupuncture for HVS. In this trial acupuncture was used as an adjunct to standard physiotherapy (in the form of breathing retraining (BR)). Study 2 was a trial to examine the feasibility of using ear acupuncture as an adjunct to physiotherapy (in the form of pulmonary rehabilitation for COPD). Hypotheses were tested using anxiety data from the Hospital Anxiety and Depression Scale. The findings from both studies suggest that it is feasible to use acupuncture as an adjunct to physiotherapy in these patient groups. Within each trial, there were no statistically significant differences in anxiety between groups at outcome. However, following the interventions, there were clinically relevant reductions in anxiety in the acupuncture groups within both studies. In study 1 the reduction in mean anxiety scores in the acupuncture group took them to below the cut-off for clinically relevant anxiety. There were also statistically significant between-group differences in breathlessness associated with HVS. These findings indicate that both body acupuncture and ear acupuncture are feasible in patients with chronic respiratory disorders, when delivered as an adjunct to physiotherapy. They also suggest that acupuncture may have clinically significant benefits for anxiety associated with respiratory disorders, as well as for symptoms such as breathlessness. This knowledge will provide practitioners with some supportive evidence for the use of acupuncture within their clinical practice.

Nontypeable Haemophilus influenzae (NTHi) is a component of the normal lung microbiome, but is also highly associated with respiratory tract infections and exacerbations of major chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD). It is not known how commensal bacteria can become pathogenic and cause inflammation in the lung, but is likely due to a breakdown in local immunity. T cell immunity may be key to controlling NTHi infection, although the responses of T cells to NTHi are not well understood. Mucosal-associated invariant T (MAIT) cells are a newly-discovered subset of innate-like T cells, which may play a role in controlling NTHi, as they recognise non-peptide antigens derived from the highly conserved vitamin B2 pathway. The role of MAIT cells in lung immunity to NTHi is also unclear. The aim of this thesis was to study the cytokine and cytotoxic responses of MAIT cells to NTHi infection, comparing these responses to those of conventional T cells. The antigen presentation and co-stimulatory mechanisms that regulate MAIT cell activation have also been explored. Conventional T cell and MAIT cell responses to NTHi were investigated using a human ex vivo lung tissue explant model and an autologous monocyte-derived macrophage (MDM)-T cell co-culture model. Cytokine and cytotoxic responses were measured by a combination of flow cytometry, ELISA and ELISpot. Blocking antibodies were used to determine the role of antigen presentation and cytokine signalling in conventional T cell and MAIT cell activation. Lung MAIT cells significantly upregulated the cytokines; IFNγ, IL-17a and TNFα, and the cytotoxic markers; granzyme B and CD107a, following NTHi infection. A greater proportion of MAIT cells were active compared to conventional T cells. In the blood-derived MDM-T cell co-culture, IFNγ expression by MAIT cells was dependent on MR1 antigen presentation and IL-12 signalling in a time-dependent manner. Cytotoxic responses were regulated by MR1 but also by a novel mechanism where IL-12 and IL-7 signalling synergised to induce granzyme B expression by upregulation of the IL-12 receptor. In contrast, conventional T cell responses predominantly relied on antigen presentation for activation. MAIT cell responses were also impaired by treatment with corticosteroids, which are commonly used to manage inflammation in chronic lung diseases, but are associated with a higher risk of developing pneumonia in COPD patients. Overall, the data in this thesis provide evidence for a role for MAIT cells in controlling NTHi infection in the lung and also highlight key differences in the regulation of innate and adaptive T cells. A further understanding of the mechanism underpinning T cell responses to NTHi infection may yield new therapeutic opportunities and improve the outcome for patients with respiratory diseases.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterised by excessive extracellular matrix (ECM) deposition and disruption of lung architecture. Wnt1-inducible signalling protein1 (WISP-1) is a matricellular protein reported to play a role in aberrant epithelialmesenchymal crosstalk in fibrotic disease. Therefore, it was hypothesised that WISP-1 contributes to pro-fibrotic changes in pulmonary fibroblasts and epithelial cells, and that these effects are mediated by alternatively spliced WISP-1 variants. Methods: MRC5, A549 and primary parenchymal fibroblasts were stimulated with the pro-fibrotic cytokine TGFβ1 or the pro-inflammatory cytokine TNFα and mRNA expression of full length (FL) WISP-1 and its splice variants (v2/3/4) was measured. Antibodies were characterised for the detection of WISP-1 protein. Expression constructs for FL WISP-1 and v2, v3 and v4 were made in pcDNA3. HEK293T cells were transfected with WISP-1 constructs and cell conditioned medium (CM) tested on A549 and MRC5 cells. A549 cells were stably transfected with FL WISP-1. Time lapse microscopy, gene expression and cell proliferation analyses were performed. Results: TNFα induced WISP-1 mRNA expression in pulmonary epithelial (A549) and fibroblast (MRC5) cell lines. TGFβ1 induced epithelial to mesenchymal transition (EMT) and WISP-1 mRNA expression in A549 cells, and suppressed WISP-1 in MRC5 cells. In primary IPF fibroblasts, higher levels of WISP-1 mRNA were detected compared to control lung fibroblasts. Expression of WISP-1 mRNA in response to TGFβ1 and TNFα was variable between donors. WISP-1 variant specific qPCR assays showed differences in expression levels between donors, reflecting the overall WISP-1 expression. FL WISP-1 was detected in cell lysates from some IPF and control parenchymal fibroblast donors. When expressed in HEK293T cells, FL WISP-1 and v2, v3 and v4 could be detected in cell lysates. FL WISP-1 and v2 were also detected as secreted proteins. When CM from the different recombinant WISP-1 expressing HEK293T cells were applied to A549 or MRC5 cells, distinct responses were observed. FL WISP-1 CM increased endogenous FL WISP-1 expression in A549 cells, but the WISP-1 variants were without effect. In contrast, WISP-1v4 CM strongly induced FL WISP-1 and WISP-1v4 in MRC5 fibroblasts while WISP-1v2 and v3 only induced their own expression and FL WISP-1 had no effect. FL and variant WISP-1 CM did not stimulate ECM production by MRC5 cells, or EMT in A549 cells. Conclusions: WISP-1 is expressed in parenchymal fibroblasts and exists as several alternatively spliced forms. WISP-1 is overexpressed in IPF fibroblasts but is not uniformly increased following pro-fibrotic or pro-inflammatory stimulation in primary fibroblasts. WISP-1 expression is increased following induction of EMT in A549 cells however overexpression of WISP-1 did not induce EMT. The differential responses of epithelial cells and fibroblasts to CM from recombinant HEK293T cells expressing different WISP-1 variants suggests that further work is required to dissect the regulation and function of WISP-1 in relation to lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a complex chronic fibroproliferative disease of unknown aetiology where aggregates of highly active fibroblasts, termed 'fibroblastic foci', produce excessive extracellular matrix (ECM) components. On standard 2D pathologic examination fibroblastic foci are considered small, distinct lesions although in 3D they have been proposed to form a highly interconnected reticulum as the leading edge of a "wave" of active fibrosis. There is increasing recognition that the ECM produced may not simply be a consequence of fibrosis but may also contribute to fibrogenesis, however the actual changes in ECM structure and function remain poorly understood. This thesis will further study fibroblastic foci and the predominant ECM constituent, fibrillar collagen, in IPF. Fibroblastic focus morphology and inter-relationships in 3D were studied by integrated microCT and histological analyses. Collagen expression in ex vivo IPF lung tissue was characterised by biochemical, biomechanical, histological, and non-linear imaging analyses. A long term model of fibrillar collagen production by fibroblastic foci was established using parenchymal lung fibroblasts explanted from IPF or non-fibrotic lung tissue. This study demonstrates that the application of multi-modal imaging methodologies can further advance concepts of disease pathogenesis in fibrotic lung diseases. It provides novel data that in 3D fibroblastic foci are heterogeneous and of varying size and complexity, suggesting previously unrecognised plasticity. It also shows that these fibroblastic structures are independent of each other, consistent with their being the product of discrete sites of lung injury and repair. It demonstrates that in IPF posttranslational modifications of collagen, rather than increased total collagen content, strongly influence the altered mechanical properties of IPF lung tissue, and identifies upregulation of multiple lysyl oxidases as therapeutic targets. Finally, it describes a novel 3D model culture system of fibroblastic focus ECM production with utility in mechanistic and therapeutic studies of fibrotic lung diseases.

Particles with a single dimension smaller than 100 nm are called nanoparticles (NPs). There is a large amount of epidemiology that anthropogenic particles cause increased mortality, increased risk of asthma and increased incidence of lung adenocarcinoma. Inhaled NPs can reach the lung terminus, interacting with a lipid lining at the air-liquid interface (pulmonary surfactant (PSf)) and with an aqueous hydrophase beneath. Studies in serum have shown that upon contact with blood, proteins very rapidly adsorb to the particle surface, thus the particle accrues a biological identity. However, no such interaction studies have been performed in the pulmonary system, where there is a complex interplay of proteins and lipids. The interaction of NPs with PSf is poorly understood. Prior to reaching the lung epithelium, NPs must translocate through this layer. The aims of this thesis are to firstly explore the interactions of NPs with proteins and lipids in the lung and secondly investigate the toxicity and effect on the immunological action of lung cells. NPs were incubated with pulmonary lavage fluid and adsorbed proteins were analysed using state-of-the-art, high resolution, quantitative mass spectrometry. To investigate the interactions of NPs with PSf, particles were incubated with a porcine surfactant. The bound lipids were analysed by qualitative mass spectrometry. The effect of NPs and the NP-protein-corona was investigated in cell lines by a number of techniques. Unique proteins were observed on each particle type, however the main contributors contribution was from a few key proteins. There was no biophysical property of these bound proteins that could predict their affinity to a particle. Surfactant associated protein A, B and D (SP-A, B, D) were observed to be bound to the particle surface. SP-A was bound with high abundance, suggesting it is likely these particles may interfere with PSf in vivo. All particles retained lipids on their surface through binding mediated by electrostatic interactions. Aminated-polystyrene and titanium dioxide NPs were capable of interfering with PSf in vitro. There was little observed effect of the particles to induce inflammation. The key findings suggest that the protein corona does not predict particle toxicity. Any observed effect is due to the core particle chemistry, not an acquired bio-identity. The toxicity of particles observed through epidemiological surveys could be caused from the inhibition of pulmonary surfactant, not from direct toxicity of the particles themselves.

Background: Rhinovirus (RV) is a common human respiratory pathogen. In healthy people RV infection causes the development of upper respiratory tract symptoms of limited duration, whilst in asthmatic patients the symptoms may be severe, prolonged and cause exacerbation of disease. Asthmatics were reported to have dysfunctional bronchial epithelial barrier with decreased junctional protein expression and impaired innate immune responses to infection, which may contribute to symptom exacerbation. MicroRNA (miR) are small RNAs that specifically bind to the 3' untranslated regions (3'UTRs) of target messenger RNAS (mRNA) and alter protein expression by influencing mRNA stability and translation. It was hypothesised that RV infection of human bronchial epithelial cells (HBEC) alters the expression of miRNA-23a, -200b, -200c and -429-miRs which are predicted to target mRNAs encoding proteins relevant to epithelial barrier integrity and innate immunity. Hypotheses: HRV-16 infection of culture differentiated airway epithelia alters levels of miR-23a and the miR-200 family (miR-200b, -200c, -429). MicroRNA responses will be different in healthy and asthmatic patients, potentially contributing to decreased epithelial integrity and impaired innate immune responses observed in asthmatics. Methods: Primary human bronchial epithelial cells from healthy and asthmatic donors were differentiated for 21 days at an air-liquid interface (ALI) before being infected with HRV-16. Supernatants and cells were harvested at 24, 48 and 72 hours. Cells were lysed for RNA extraction and levels of miRNAs and in silico predicted targets (Zinc finger E-box-binding homeobox 1 (ZEB1), TNF-α Inhibitory Protein 3 (TNFAIP3), E-cadherin and occludin) were assessed. Supernatants were tested for interferon (IFN)-λ protein release. Differentiated HBECs were treated for 72 hours with IFN-λ to assess the effect of HRV-16 induced antiviral mediators on expression of microRNAs and target mRNA/protein in intact epithelial barrier. Differentiating HBECs were treated for 21 days with IFN-λ to assess the effect of HRV-16 induced antiviral mediators on expression of miRNAs and target mRNA/protein in differentiating, 'repairing' barrier. Additional epithelial barrier characteristics were assessed in response to IFN-λ treatment through imunoflourescent staining, cell counts, fluorescein isothocynate (FITC) 4kD dextran assays and transepithelial resistance (TER). Dual luciferase reporter assays were utilised to confirm miR-23a binding to predicted sequence target in 3'UTR of TNFAIP3. Results: MiR-200b, -429 and -23a were significantly decreased in response to HRV-16-with a trend for greater reductions in asthmatic donors. Predicted miR-23a target TNFAIP3, predicted miR-429 target occludin and E-cadherin had significantly altered mRNA in response to HRV-16. There were no alterations to ZEB1. HRV-16 lead to significant inductions of IFN-λ mRNA and protein release. IFN-λ treatment of differentiated ALI cultures showed significant reductions of miR-23a and miR-429 in cultures from healthy donors with elevated TNFAIP3 and occludin mRNA and elevated ZEB1 protein expression. In cultures from asthmatic donors, miR-23a was significantly decreased in response to IFN-λ but there were no significant reductions in miR-429 with elevated TNFAIP3 expression and elevated ZEB1 protein expression. IFN-λ treatment decreased TER and changed cell morphology with less pronounced effects in asthmatic individuals. In differentiating ALI cultures, miR-23a and miR-429 were significantly decreased in healthy individuals, but no significant alterations were observed in asthmatics. ZEB1, E-cadherin, TNFAIP3 and occludin mRNA were significantly changed in response to IFN-λ in healthy donors with significant changes to E-cadherin and TNFAIP3 observed in asthmatic donors. Western blotting results were inconclusive. IFN-λ treatment decreased TER, increased FITC passage, decreased cell numbers and changed cell morphology with less pronounced effects in asthmatic individuals. Dual reporter luciferase assays confirmed miR-23a binding to predicted target sequence in 3'UTR of TNFAIP3/A20, which may affect NF-κB activation. Conclusions: HRV-16 significantly reduces miR-200b, -429 and -23a which may account for alterations in epithelial barrier integrity and innate immune responses via microRNA targets ZEB1, E-cadherin, occludin and TNFAIP3/A20. IFN-λ protein, released in response to HRV-16, effects epithelial barrier properties in both intact and repairing barrier which may be mediated by significant reductions in miRNAs of interest and aforementioned targets. Asthmatic donors appear to be less responsive to IFN-λ which may underpin the impaired epithelial barrier and impaired innate immune responses to HRV-16 infection observed in these patients.

Smooth muscle contraction, tissue remodelling and chronic inflammation are features common to many lung conditions including asthma and pulmonary arterial hypertension (PAH). Three people in the UK die each day from a fatal asthma exacerbation, whilst right heart failure caused by increased resistance in the pulmonary arteries is an end-point that over half of patients with PAH can expect just five years after diagnosis. Novel relaxants to address the inappropriate smooth muscle contraction in these diseases are required. Recently-identified families of specialised pro-resolving lipid mediators (SPMs) including resolvins (Rv), lipoxins (LX), protectins (PD) and maresins actively limit the acute inflammatory response through the activation of G-protein-coupled receptors, but their roles in smooth muscle contraction are poorly understood. The aims of this thesis were: to determine whether SPMs, in particular RvD1, RvE1, LXA4 and PDX, can modulate spontaneous or growth factor-induced contraction of cell-populated collagen lattices; to use wire myography to define whether Rvs D1, D2 and E1 can modulate contractility in bronchiole and artery segments from rat and human donors; and finally to use immunohistology to localise expression of the pro-resolving receptors ChemR23, GPR32 and ALX in human pulmonary artery (HPA).None of the SPMs investigated affected spontaneous contraction of collagen lattices populated with human lung fibroblasts (HLF), human bronchial smooth muscle cells (HBSMC) or human umbilical artery smooth muscle cells (HUASMC), nor did they affect TGFβ1-enhanced lattice contraction. However, RvE1 abolished the platelet-derived growth factor (PDGF)-enhanced contraction of HBSMC lattices (p < 0.05). In addition, PDX weakly inhibited contraction of lattices populated with HLF or HBSMC, while RvD1 and LXA4 had no effect. Wire myography was used to investigate the ability of RvD1, RvD2 and RvE1 to prevent or reverse contraction of bronchiole segments isolated from rat and human lung. None of the resolvins tested were able to reverse carbachol (CCh)-induced pre-constriction in rat bronchioles or leukotriene D4 (LTD4)-induced preconstriction in human bronchioles. Pre-treatment with resolvins D1 or E1 failed to prevent subsequent contraction of rat bronchioles induced by either CCh or the stable thromboxane mimetic U46619, compared to the same segments contracted previously without resolvin. Pre-treatment of rat and human bronchioles with RvD1 (10 nM) appeared to dampen LTD4-induced constriction, although complications with suspected tachyphylaxis and generally low responsiveness to LTD4 in rat bronchioles may have precluded significant outcomes. Wire myography was used to explore Rv effects in rat and human blood vessels. Rvs D1, D2 and E1 all failed to reverse phenylephrine (PE)- or U46619-induced pre-constriction in segments of rat thoracic aorta (RTA), and HPA, these resolvins did not reverse pre-constriction induced by LTD4. Pre-treatment of HPA with RvD1 or RvE1 had no effect on subsequent constriction by U46619, while their effects on LTD4-induced constriction were masked by changing baseline responses to this contractile agonist, as seen in bronchiole segments. However, in naive segments of RTA pre-treated for 1 or 24 hour with or without resolvin in parallel, RvE1 (0.1-300 nM), RvD1 and RvD2 (1-100 nM) each significantly inhibited constriction to U46619 with a bell-shaped inhibition curve, when compared to the control segments constricted by U46619 without resolvin (10 nM RvE1 p < 0.0001; 10 nM RvD1 p < 0.01; 10 nM RvD2 p < 0.05). The significant inhibitory effect of RvE1 (1 hour) was also seen in U46619-constricted HPA segments (p < 0.0001 with 10 nM RvE1), but not in RTA constricted with PE, indicating a selective effect on thromboxane receptor signalling. Immunohistochemistry of paraffin wax-embedded HPA sections indicated that the pro-resolving receptors GPR32 and ChemR23 were both expressed in the vascular smooth muscle, with GPR32 also apparent in the endothelium. This thesis provides the first evidence that acute pre-treatment with SPMs can directly impair the contractile responses of intact segments of rat aorta and human pulmonary arteries to well-known contractile agonists, and that they can also modulate the PDGF-enhanced contraction of 3D collagen matrices by human bronchial smooth muscle cells. These findings provide novel insights into SPM activity with important implications for understanding the regulation of smooth muscle contractility in chronic inflammatory diseases such as asthma, PAH and systemic hypertension.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease. Pathogenesis involves the aberrant accumulation of myofibroblasts and excessive deposition of matrix components. Its aetiology is unknown and treatment options are limited. The histone deacetylase inhibitor FK228 has been shown to have anti-proliferative effects on fibroblasts, and therefore may be a candidate for IPF therapy. This thesis describes the in-depth proteomic characterisation of fibroblasts from patients with IPF, and their response to FK228. Comparison of protein expression between IPF and non-IPF fibroblasts revealed over 120 aberrantly expressed proteins in this disease, including those involved in transcriptional regulation, which have the potential for use as diagnostic or monitoring biomarkers. Extensive validation is now required for their translation to the clinic. IPF biomarkers are greatly sought after as there are currently none in clinical use. FK228 treatment caused a decrease in IPF fibroblast metabolic activity and proliferation through cell cycle arrest, and significantly affected the expression of hundreds of proteins. The proteomic responses of IPF fibroblasts grown in 2D and 3D culture were shown to differ substantially; there was little difference in the number of proteins quantified between culture systems, but treatment affected the expression of different proteins. This highlights the importance of culture system selection for studying IPF and for testing therapeutics effectively in vitro. The method for profiling cultured fibroblasts using proteomics was firstly developed in the MRC-5 cell line, to optimise cell lysis, protein isolation and digestion, and mass spectrometry analysis for global protein identification. Intracellular and extracellular proteomic analysis of these cells following optimisation, with and without transforming growth factor beta-1 (TGF-β₁) stimulation is also discussed in this thesis. This dataset provides to the author's knowledge the most comprehensive characterisation of the fibroblastic response to this profibrotic cytokine to date, with more than 5000 proteins quantified. Data analyses highlighted biological processes that may be dysregulated in fibrotic disease, including cell cycle regulation and cellular organisation. Finally, the first steps in the development of a method using immunoprecipitation enrichment to study the IPF fibroblast acetylome in response to FK228 are described, for the identification of off-target effects. Fewer than 100 acetylated peptides were identified by mass spectrometry analysis, a considerably lower number than found in previously published reports, highlighting the inefficiency of the technique for global enrichment of acetylated peptides in this study.

Respiratory tract infections (RTI) are responsible for over 4 million deaths per year worldwide. Microbial carriage in the upper respiratory tract is a precursor to respiratory infection and facilitates person-to-person transmission. A large community-based swabbing study was conducted, enabling the collection of a large number of swab samples that would provide key information concerning the epidemiology and ecology of respiratory tract communities. Traditional culture-based techniques, molecular methods and ecological and mathematical modelling methods were used. Participation of members of the community within the swabbing study was shown to be greater within the self-swabbing group, in older individuals and in less deprived locations. Carriage of bacterial and viral species within the respiratory tract was shown to vary with participant age, recent RTI and the presence of other species. Self-taken swabs were largely non-inferior to healthcare professional (HCP)-taken swabs in assessing carriage of the targeted bacteria, offering a cheaper and more flexible alternative to HCP swabbing. Large numbers of capsular types (serotypes), sequence types and low levels of vaccine-targeted types demonstrate the genetic diversity of respiratory bacteria as well as their evolution in response to immunisation. Microbial respiratory community structure was shown to be highly variable with less nested communities and facilitative relationships between species within young individuals and those with recent RTI potentially enhancing transmission and survival of carried species. Neutral and niche processes were both found to be important in respiratory tract community assembly. These insights into respiratory tract communities will allow predictions of microbial variation as a result of infection, varying age and season. Future work will involve 16S rDNA community analysis, further development of ecological methods and the conduction of larger multi-centre carriage studies.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible disease of the lung, characterised by excessive extracellular matrix (ECM) deposition and loss of pulmonary function. Fibroblasts are the main cell type responsible for ECM deposition and peptides from the renin-angiotensin system (RAS) have been shown to modulate fibroblast function. Angiotensin (ANG)-II, has been shown to exert profibrotic effects on human cardiac fibroblasts, such as increased collagen deposition. ANGII is known to bind to two receptors (AT1R and AT2R), with profibrotic effects attributed to AT1R. ANGII is cleaved to form ANG(1-7). This peptide has been shown to exert antifibrotic effects on fibroblasts, such as decreased cell proliferation, via binding to the Mas receptor. However, most investigations have been performed in rat fibroblasts. Another related peptide called ANG(1-9), has also been shown to exert antifibrotic effects on fibroblasts, via binding to AT2R, again this work has only been performed in rat fibroblasts. The main aims of this thesis were to investigate the effects of ANGII and related peptides on signalling pathways in both IPF derived and healthy human lung fibroblasts (HLFs) and to explore the effects of ANG peptides on HLF function. A second aim was to characterise Mas receptor expression on HLFs. The effects of ANGII, ANG(1-7) and six related peptides on intracellular Ca2+ release were explored and the ability of ANGII to phosphorylate ERK was also investigated. Fibroblast functions in response to both ANGII and ANG(1-7) were also examined, including proliferation, collagen deposition, matrix metalloproteinase (MMP) and cytokine secretion. Receptor involvement was investigated with telmisartan (AT1R antagonist), PD-123319 (AT2R antagonist) and A-779 (Mas receptor antagonist). Mas receptor expression on HLFs was also investigated with immunocytochemistry and radioligand binding studies. ANGI ANGII and ANGIII caused intracellular Ca2+ release via AT1R. ANGII also induced ERK phosphorylation, although this did not translate to fibroblast function. No effects of ANGII were observed on cell proliferation, collagen deposition, MMP secretion or IL-6 production. In the Ca2+ assay, ANG(1-7) and ANG(1-9) partially inhibited the responses to both ANGII and ANGIII. These effects did not appear to be modulated by AT2R or the Mas receptor, indicating a novel binding site for these two peptides. The effects of ANG(1-7) did not appear to correlate with functional activity, with no reduction in cell proliferation or collagen deposition observed with this peptide. Immunocytochemistry showed clear nuclear expression of the Mas receptor in HLFs, with very low cell surface expression, providing an explanation for the lack of functional response. Together these results indicate that although many ANG peptides can affect intracellular signalling pathways, peptides from the RAS do not affect functional responses of HLFs.

The primary aim of this thesis was to present the development of the prototype of a theory-based web intervention aiming to support decision-making of the general adult population on whether to seek professional help or self-care for acute respiratory tract infections. The thesis comprised of a systematic review, two qualitative studies, one survey, and a step-by-step development of the intervention. All the empirical studies aimed to identify the theoretical and technical components that could improve the intervention. The systematic review of 18 trials aimed to identify the effectiveness of health educational materials in improving health outcomes for minor ailments such as physical health and health service use. There was mixed evidence regarding the factors that influence primary care consultations; providing educational information outside consultation appeared to be most effective. The piloting of the intervention elicited feedback, via 21 interviews, regarding the content of the intervention e.g. reducing the length of the information, making screening questions clearer, and clarifying when they need to seek professional help. Comments about the format included improving the navigation and aesthetics of the materials by adding more pictures and colour as well as reducing the use of jargon language. The second qualitative study aimed to elicit the underlying reasons to consult a clinician. Findings indicated that consultation was linked to uncertainty about the symptoms, severity and the impact on everyday activities, and past antibiotic prescribing. The theories closely identifying with the arising constructs were Social Cognitive theory, the Common Sense of Illness Representations, and the Beliefs about Medication. The last study of thesis, a survey of 323 participants, showed that the most significant factors linked to the decision not to seek professional help were strong beliefs that symptoms can resolve on their own and seeking help from the pharmacist prior seeing the GP. The findings from the empirical studies contributed further into the development of the intervention as a new resource to help individuals decide whether to seek professional help or self-care for their symptoms. Further work for the online version of the intervention, including tailoring of theoretical factors and including more representative sample, can enhance its validity and effectiveness.

Asthma is one of the commonest chronic diseases worldwide and severe asthma sufferers experience recurrent exacerbations. Exacerbations are predominantly virus-induced and have been linked to defective interferon responses by airway immune and structural cells. Ascertaining the molecular mechanisms underlying this deficiency is a major research goal in order to identify new therapeutic targets. We hypothesised that impaired interferon responses in severe asthma are caused by aberrations in microRNA expression in alveolar macrophages. MicroRNAs are non-coding RNA molecules that down-regulate gene expression. We identified and focused on 3 microRNAs predicted to target Toll-like receptor 7 (TLR7). We hypothesised that reduced expression of TLR7 would lead to reduced innate immune responses to the virus and that manipulating the expression of these microRNAs could restore the defective interferon response in asthmatic alveolar macrophages. Alveolar macrophages were isolated from bronchoalveolar lavage from healthy and severe asthma subjects. Expression of microRNAs miR-150, -152 and -375 was increased and expression of TLR7reduced in alveolar macrophages from severe asthma subjects compared to healthy subjects. Using a TLR7- luciferase reporter construct we showed that the 3 microRNAs directly targeted the 3’UTR of TLR7 and operated synergistically to reduce its expression. Reduced TLR7 expression was association with impaired interferon responses to rhinovirus and imiquimod, a specific TLR7 agonist, and correlated inversely with number of disease exacerbations. Ex vivo knock-down of these microRNAs restored TLR7 expression with concomitant augmentation of virus-induced interferon production. In conclusion, the results presented here show that increased expression of miR-150, miR-152 and miR-375 in severe asthma leads to reduced expression of TLR7 in alveolar macrophages and contributes to the impaired innate immune response to rhinovirus. This would certainly predispose the individual to more frequent and prolonged exacerbations due to reduced viral clearance by airway cells. Importantly we show that knock-down of these microRNAs in alveolar macrophages rescues the expression of interferon, providing a novel therapeutic target for the prevention and treatment of asthma exacerbations.

Introduction: Asthma is a global burden, leading to around 180,000 deaths worldwide annually. Translation of therapies from animal models is limited, suggesting these models do not recapitulate adequately genetic and environmental aspects of asthma. Existing human cell culture models are usually simplistic and do not mimic the in vivo tissue architecture; therefore there is a need to improve three-dimensional human tissue culture models. We hypothesised that use of cell sheet engineering would help overcome this problem. Aims: Thermoresponsive polymers or ultrasound standing waves (Sonotweezers) will be tested for their compatibility for the formation of cell sheets which can be used for tissue engineering. Methods: Poly (2-alkyl-2-oxazolines) are polymers with thermoresponsive properties and were tested for biocompatibility using cell motility and adhesion assays, immunofluorescent staining, and measurement of p38 phosphorylation. Multiple cell types were seeded onto thermoresponsive polymers (either NiPAAm, poly(2-alkyl-2-oxazolines) or thermo-gels of poly(2-isopropyl-2-oxazoline)-carboxymethylcellulose)) and cell sheet release assessed after incubation at 20ºC. Cell sheets were also created using ultrasound standing waves (Sonotweezers) or incubation on a thermo-gel. Adhesion junctions in cell sheets were assessed by staining for E-cadherin, ZO-1 and the actin cytoskeleton; cell viability was monitored using 7-Aminoactinomycin D. Subsequently, epithelial cell sheets were used to created co-cultures with fibroblasts. Cytokine release (IL-6 and IP-10) following Poly (I:C) stimulation from cell-sheet co-cultures was compared to conventional models by ELISA. Results: Using conventional cell culture techniques a multi-layered cell structure by the addition of epithelial cells onto a confluent fibroblast layer was not possible as it resulted in redistribution of the cells to form a single layer comprising islands of epithelial cells surrounded by fibroblasts. Consequently alternative methods were explored using thermoresponsive polymers or Sonotweezers. Cells could be lifted from a commercial NiPAAm coated dish (UpCell) after attachment to a membrane, but during the release process cells either rounded up and lost contact (fibroblasts, HeLa cells) or epithelial cell sheets were damaged and incompletely released. Sonotweezers also could not generate sufficient force to release and levitate the cells. As alternatives, cell sheets were created by levitation in the Sonotweezers device or overlaying the cells on a thermo-gel to allow cell sheet formation followed by sedimentation onto an underlying layer of fibroblasts. Levitation of single cells resulted in the formation of a cell sheet within 2 hours, which gradually contracted becoming three-dimensional by 24 hours. Contraction could be inhibited by removal of Ca2+ to prevent adherens junction formation or by adding cytochalasin D to prevent actin filaments or an E-cadherin neutralising antibody to prevent adherens junction formation. After 2 hours of levitation, the cell sheet could be placed onto confluent MRC5 fibroblasts and epithelial cells used plithotaxis to spread across the fibroblasts to create two distinct layers. Oxazoline polymers with a range of hydrophobicities covalently attached to glass were biocompatible, but not thermoresponsive. A gel of poly(2-isopropyl-2-oxazoline-co-2-butyl2-oxazoline)-carboxymethylcellulose was thermoresponsive, enabling formation of epithelial cell sheets which were used to form cell-sheet co-cultures. The cell-sheet co-cultures achieved an electrically tight barrier and when challenged with the viral mimic Poly(I:C), showed increased IL-6 and IP-10 release. IL-6 release was predominantly apical, whereas IP-10 was basolateral, suggesting polarised mediator release. Conclusions: Multi-layered cell culture models can be created using either the Sonotweezers device or gelling polymers. The latter offers potential for formation of multi-layered structures in a high through-put manner.

Asthma is a heterogeneous chronic inflammatory disease characterised by recurrent, reversible airflow obstruction, bronchial hyperresponsiveness (BHR) and airway remodelling. Impaired lung function in childhood asthma has been associated with polymorphisms in ADAM33. Soluble ADAM33 (sADAM33) is increased in bronchial lavage fluid (BALF) from asthmatics and is inversely correlated with FEV1. sADAM33 is induced by TGF-β present in asthmatic lungs, and promotes angiogenesis. ADAM33 expression is developmentally regulated and is influenced by maternal allergy via IL-13. This thesis will examine the hypothesis that alteration in the expression of ADAM33 will influence lung structure, affecting vessel and smooth muscle formation and these subsequent changes will impact on pulmonary function in airway inflammation. In DOX inducible Il-13 transgenic mice, significant neutrophilic inflammation and goblet cell metaplasia were observed. BHR was induced after methacholine challenge and IHC showed increased bronchial smooth muscle. Similarly, in human embryonic and juvenile mouse lungs, IL-13 suppressed Adam33 mRNA but not Acta2. ADAM33 was identified in BALF of the overexpressing mice. ADAM33 enzymatic activity was also significantly increased. In the ADAM33 transgenic model, induction of ADAM33 resulted in enzymatically active sADAM33 in BALF. ADAM33 significantly increased the expression of fibrotic markers suggesting regulation of pulmonary myogenesis, fibrogenesis, and angiogenesis. Consistent with this, immunofluorescence revealed airway remodelling with increased smooth muscle, collagen deposition and vessel formation in ADAM33 mice. In contrast, inflammatory cell counts in BALF, expression of inflammatory mediators and mucus related genes were not altered by ADAM33. In Adam33 -/- challenged with HDM, there was less BHR and eosinophilic and neutrophilic inflammation compared to Adam33 +/+. HDM caused suppression of Adam33 mRNA as well as ectodomain shedding of ADAM33 protein in BALF of wildtype mice that was absent in Adam33 -/-. The study provides novel data showing expression of sADAM33 in airways to induce myogenesis, fibrogenesis and angiogenesis, consistent with a process causing airway remodelling in the absence of inflammation.

Background: More than half of asthma exacerbations are caused by infection with human rhinovirus. When infected with rhinovirus (RV), macrophages produce tumour necrosis factor α (TNF-α), a pro-inflammatory cytokine that has been reported to be increased in the bronchoalveolar lavage of severe asthmatics. MicroRNAs thought to be involved in inflammatory responses were seen to be upregulated in alveolar macrophages of asthmatic patients. Hypothesis: MicroRNA profile is altered in asthmatic macrophages and airway and this leads to an exaggerated pro-inflammatory response to rhinoviral infections. Results: MiR-27a, miR-155 and miR-152 contributed to RV-induced TNF-α overexpression in alveolar macrophages. Upregulation of these microRNAs had an effect not only the cells' total RNA content but also polysome-bound genes, which only marginally overlapped with Targetscan 6.2 predictions. Only 24 of these genes were common to total and polysome-bound RNA. The microRNA and inflammatory profiles of these cells could be affected by the contents of bronchoalveolar lavage, in particular exosomes. Only 6% of exosomal RNA from severe asthmatic BAL is composed of microRNAs, compared to 27% in healthy controls. Dysregulated microRNAs could affect relevant pathways such as epithelial barrier, transforming growth factor β (TGF-β) and mitogen-activated protein kinases (MAPK) pathways, among others. Conclusions: MiR-27a, miR-155 and miR-152 are upregulated in macrophages from asthmatic patients. These microRNAs are able to increase TNF-α production following RV infection which could lead to worsening of symptoms in an asthmatic exacerbation. Such dysregulation could arise from differential loading of exosomes by airway cells. In order to improve their efficacy, microRNA function studies should ally in silico prediction tools with sequencing of both total and polysome-bound RNA.

T-cells are key orchestrators of airways inflammation, but the relative roles of different human T-cell subsets remain unclear. The aim of my PhD was to carry out a detailed investigation of T cell phenotypes in asthma in relation to severity and virus-induced exacerbations, with particular focus on interleukin-17 and TH 17 cells, and the recently described mucosal associated invariant T (MAlT) cells, to improve characterisation of severe asthma versus milder forms of asthma. A role for interleukin-17 secreting TH17 cells in asthma has been suggested by several groups. I used clinical and physiologic phenotyping to compare T-cell subsets in health and a spectrum of different asthma severities. Samples obtained via sputum induction, phlebotomy, and bronchoscopy were phenotyped using 9-colour flow-cytometry/sorting, RT-qPCR and multiplex ELlSA. The results of my thesis confirm the pre-eminence of TH2 cells in asthma and provide further evidence of a deficiency of bronchoalveolar Treg in severe asthma, as well as new evidence of a role for CD8+ Tc2 cells in eosinophilic disease. Conversely, the data do not indicate a significant role for TH17 or yo-17 cells in asthma. Mucosal immunity is intrinsically linked to the associated commensal or pathogenic microbes. In an exploratory study of these interactions I employed deep-sequencing to characterise the whole microbial and viral metagenome of the airways in asthma and health. MAlT cells are novel innate-like T-cells which express an invariant TCRa chain and recognise the highly-conserved restriction molecule MR1. I observed a selective deficiency of MAlT cells in asthma, which was not related to age, but exacerbated by systemic corticosteroids and subject to seasonal variation, indicating their possible regulation by vitamin D. I established MAlT cell-lines and observed heterogeneity of cytokine expression profiles. These findings open exciting new avenues for research in this emerging area of T cell biology.

Rationale: A key factor involved with the progressive decline in lung function in severe asthma is airway remodelling. This involves collagen deposition within the lamina reticularis, matrix deposition in the submucosa, smooth muscle hyperplasia, and microvascular and neuronal proliferation. Aims: The main aim of this thesis was to test the hypothesis that epithelial susceptibility to environmental injury and a prolonged tissue repair response result in activation of the epithelial mesenchymal trophic unit (EMTU) that in turn promotes airway wall remodelling. Methods: Investigation of epithelial mesenchymal signalling involved use of cells derived from normal or asthmatic volunteers and cultured in vitro. These were tested in simple monocultures and then a „tissue engineered‟ construct was developed to enable assessment of responses in a complex cell model that closely mimics the human bronchial mucosa. Results: Infection of asthmatic bronchial epithelial cells (AS PBEC) with RV-16 caused significantly less apoptosis when compared to infection of bronchial epithelial cells obtained from healthy controls (HC PBEC). As a consequence of this RV replicated more in AS PBEC; this defect may explain the higher susceptibility of the lower airways to RV infection in asthmatic subjects. Using primary bronchial fibroblast cultures, collagen type I and fibronectin significantly increased adhesion rates, while laminin reduced differentiation of fibroblasts into myofibroblasts. None of the ECM components that were evaluated affected fibroblast survival. Although asthmatic fibroblasts expressed more αSMA than normal fibroblasts, no significant difference in terms of fibroblast differentiation was found when different ECM components, of relevance to asthma pathogenesis, were studied. When bronchial fibroblasts were exposed to conditioned media obtained from PBEC infected with RV16, there was no effect on markers of remodelling but there was a marked amplification of the epithelial inflammatory response, mainly due to increased release of IL8, IL6, RANTES and IP-10, suggesting that during a virus-induced exacerbation the fibroblasts promote inflammation. To investigate susceptibility to oxidant stress, PBEC were treated with 20% cigarette smoke extract (CSE). AS PBECs were more susceptible to CSE with a significant increase of early apoptotic (EA) cells compared to HS PBEC. Reduced glutathione protected both HS and AS PBEC from CSE- induced cell death causing a significant increase in cell viability, with a concomitant decrease in apoptosis. Oxidative stress-induced apoptosis in PBECs did not follow the canonical caspase pathways, but rather depended on a more direct mitochondrial damage pathway. The ECM also appeared to play a role in oxidative stress-induced cell death, with collagen IV being most effective in reducing H2O2-induced apoptosis. Finally, we developed and characterised a novel 3D in vitro model of human bronchial mucosa that proved to be very similar to the normal in vivo counterpart with a well differentiated epithelial layer composed of ciliated and goblet cells producing mucus and supported by a functional basement membrane which separated the epithelium from a mesenchymal layer containing fibroblasts dispersed in a well organised ECM. This model allowed investigation of the long term effects of CSE exposure which showed that CSE caused many characteristic signs of remodelling such as thickening of the basement membrane, disarray of the ECM, loss of ciliated cells and hyperproduction of mucus. These effects occurred in the absence of inflammatory or immune cells. Conclusions: The finding that epithelial cells from asthmatic donors have increased susceptibility to viral and oxidative stress emphasises the importance of tissue susceptibility in asthma. These findings, together with the observations that there is substantial cross talk between epithelial cells and fibroblasts supports the concept that abnormal function of the EMTU is an important contributory factor for asthma pathogenesis.

The collectins surfactant protein A (SP-A) and SP-D are found in the lining fluid of the lung and form an important component of innate pulmonary defenses; they consist of a N-terminal region, a collagenous region, a neck region and a carbohydrate recognition domain (CRD). SP-A and SP-D bind to and have antiviral properties in vivo against the enveloped viruses respiratory syncytial virus (RSV) and influenza A virus (IAV). A recombinant fragment of SP-D (rfhSP-D) has also been developed and is composed of the neck, CRD and a short collagenous region of SP-D. The rfhSP-D retains the ability to bind to RSV and IAV. Human rhinovirus (HRV) is a non-enveloped virus and is the predominant cause of the common cold. HRV can also cause exacerbations in patients with chronic airway disease. Based on the known antiviral effects of SP-A and SP-D, and their localisation in the upper and lower airways, the present thesis sought to investigate the research question 'Can SP-A, SP-D and rfhSP-D bind to and inhibit infectivity of HRV?' Methods to purify and detect HRV were established and used in experiments with SP-A and SP-D purified from bronchoalveolar lavage and rfhSP-D expressed in Escherichia coli. A novel interaction between SP-D and rfhSP-D with HRV was identified using co-irnmunoprecipitation and surface plasmon resonance; no such interaction was detected with SP-A. Binding was calcium dependent and inhibited by the presence of sugars, indicating that binding is via the CRD. Monolayers of HeLa cells, 16HBE cells or undifferentiated primary nasal epithelial cells were infected with HRV coincubated with collectins and the infection level was assessed using flow cytometry. The presence of collectins did not significantly alter the infection level. This is the first report of a component of the innate immune system binding to the surface of HRV.

Crackle sounds are associated with a variety of lung disorders. Smoking is also associated with many of the changes in the lung and airways leading to crackles. However, studying crackles as an indication of pathologic changes related to cigarette smoking in the lung is an underdeveloped area of research which needs to be explored. This study was undertaken to investigate whether differences in the crackles' characteristics (duration of two cycle deflection (2CD) and number of crackles per breathing cycle (NCBC)) in the lung of smoking and non-smoking young adults could be found and to quantify these differences, if present, using a digital stethoscope and computer aided lung sound analysis (CALSA). Sixty male subjects (30 smokers and 30 non-smokers) with an average age of 26.6 years (SD ± 4.7) were recruited, drawn from students at the University of Southampton in the United Kingdom. The lung sound data were recorded on one occasion using a digital stethoscope connected to a laptop running MATLAB to record and store the lung sounds from seven anatomical sites on the chest. The 2CD and NCBC per site in 25 second recordings were calculated using data from each of the anatomical sites used for recording lung sounds (excluding the trachea). No statistically significant differences in NCBC per site were found between smokers and non-smokers at any anatomical location. The 2CD per site data revealed some statistically significant differences at both anterior sites (anterior left: F (2, 57) = 9.40, P = 0.00; anterior right: F (2, 57) = 9.51, P = 0.00)) and both lateral sites (middle left: F (2, 57) = 4.2, P = 0.02; middle right: F (2, 57) = 4.36, P = 0.02)). The hypothesis that lung crackle’s 2CD differ between smokers and non-smokers has been supported but the hypothesis that NCBC differ between smokers and non-smokers has not been supported.

Communication often takes place in reverberant spaces making it harder for listeners to understand speech. In such difficult environments, listeners would benefit from being able to locate the sound source. In noisy or reverberant environments hearing-aid wearers often complain that their aids do not sufficiently help to understand speech or to localise a sound source. Simple amplification does not fully resolve the problem and sometimes makes it worse. Recent improvements in hearing aids, such as compression and filtering, can significantly alter the Interaural Time Difference (ITD) and the Inter-aural Level Difference (ILD) cues. Digital signal processing also tends to restrict the availability of fine structure cues, thereby forcing the listener to rely on envelope and level cues. The effect of digital signal processing on localisation, as felt by hearing aid wearers in different listening environments, is not well investigated. In this thesis, we aimed to investigate the effect of reverberation on localisation performance of normal hearing and hearing impaired listeners, and to determine the effects that hearing aids have on localisation cues. Three sets of experiments were conducted: in the first set (n=22 normal hearing listeners) results showed that the participants’ sound localisation ability in simulated reverberant environments is not significantly different from performance in a real reverberation chamber. In the second set of four experiments (n=16 normal hearing listeners), sound localisation ability was tested by introducing simulated reverberation and varying signal onset/offset times of different stimuli – i.e. speech, high-pass speech, low-pass speech, pink noise, 4 kHz pure tone, and 500 Hz pure tone. In the third set of experiments (n=28 bilateral Siemens Prisma 2 Pro hearing aid users) we investigated aided and unaided localisation ability of hearing impaired listeners in anechoic and simulated reverberant environments. Participants were seated in the middle of 21 loudspeakers that were arranged in a frontal horizontal arc (180°) in an anechoic chamber. Simulated reverberation was presented from four corner-speakers. We also performed physical measurements of ITDs and ILDs using a KEMAR simulator. Normal hearing listeners were not significantly affected in their ability to localise speech and pink noise stimuli in reverberation, however reverberation did have a significant effect on localising a 500 Hz pure tone. Hearing impaired listeners performed consistently worse in all simulated reverberant conditions. However, performance for speech stimuli was only significantly worse in the aided conditions. Unaided hearing impaired listeners showed decreased performance in simulated reverberation, specifically, when sounds came from lateral directions. Moreover, low-pass pink noise was most affected by simulated reverberation both in aided and unaided conditions, indicating that reverberation mainly affects ITD cues. Hearing impaired listeners performed significantly worse in all conditions when using their hearing aids. Physical measurements and psychoacoustic experiments consistently indicated that amplification mainly affected the ILD cues. We concluded that reverberation destroys the fine structure ITD cues in sound signals to some extent, thereby reducing localisation performance of hearing impaired listeners for low frequency stimuli. Furthermore we found that hearing aid compression affects ILD cues, which impairs the ability of hearing impaired listener to localise a sound source. Aided sound localisation could be improved for bilateral hearing aid users, if the aids would synchronize compression between both sides.

Tryptases, the dominant secretory granular proteins from human mast cells, are emerging as important mediators in asthma and allergy. The β- and α- tryptases have highly similar nucleotide sequences and located on the same locus. While the entire population expresses β-tryptase, the α-tryptase gene exhibits copy number variation (CNV). We have studied the association of expression of these allelic variants with asthma or allergic diseases. We have investigated also the potential actions of β- and α-tryptases in vitro and in vivo. We have found that the one alpha tryptase copy allele was significantly associated with lower total serum IgE levels (Z= -2.39, p=0.01) and a tri-allelic architecture with alleles carrying no, one or two copies of the α-tryptase gene was postulated. The addition of βtryptase to epithelial cells induced upregulation of mRNA for IL-8, IL-6 and TNF-α, while α-tryptase on the other hand was without effect in this model. Injection of β-tryptase into the mouse peritoneum induced great accumulation of neutrophils but accumulation of other cell types was less marked. Under the same conditions, injection of α- tryptase induced less neutrophilia but eosinophils, macrophages and mast cells numbers were significantly increased. The actions of β-tryptase seemed be independent of PAR-2 receptors but not the case for α-tryptase, where PAR-2 pathway might take the leads. In conclusion, recombinant α-tryptase may be a stimulus for the recruitment of inflammatory cells and altered cytokine gene expression with effects distinct from those of β-tryptase.

Chronic obstructive pulmonary disease (COPD) is a debilitating disease that as of yet has no cure and therapy is limited to symptomatic relief. A major risk factor for the development of COPD is smoking, although the disease does have some component of genetic predisposition. In excess of £500 million funding per year is required to accommodate the needs of COPD patients and approximately 27, 000 deaths per year in the UK can be attributed to COPD. COPD is comprised of three conditions-chronic bronchitis, bronchiolitis and emphysema and these will be present in the COPD patient to varying degrees. It is well accepted that COPD is a disease characterised by increases in inflammation and as such inflammatory stimuli, such as cigarette smoke and lipopolysaccharide (LPS) from bacterial cell walls, have been associated with disease development and progression. Consequently, there are associated increases in inflammatory cytokines such as TNFα in the COPD patient. Although inflammation plays a major role in the pathogenesis of COPD there are other factors to consider such as proteolytic damage caused by disturbances in the proteinase/anti-proteinase balance. This is of particular importance in emphysema where increases in neutrophil elastase concentration results in the destruction of elastin fibres which results in a decrease in lung function. Elastase is also known to contribute to the mucus hypersecretion associated with chronic bronchitis. The proteolytic actions of elastase are well characterised but there is gathering evidence to suggest that it may also be able to act as an inflammatory stimulus, thereby increasing its role in the pathogenesis of COPD. This study has utilised a human lung explant model to investigate whether elastase can initiate an inflammatory response; concentrations of the pro-inflammatory cytokine TNFα and the anti-inflammatory cytokine IL-10 in the culture supernatant have been investigated as part of this. Data from this model (n=36) has shown that elastase can significantly increase both TNFα and IL-10 compared to control. Elastase stimulation, for 24hrs, caused the release of 30.1±8.0pg TNFα/mg tissue and 3.1±0.5pg IL-10/mg tissue. This response is comparable to that produced by LPS. We have also found that elastase can induce a Th2 type response from the parenchymal explants, with increases in IL-4, IL-5 and IL-13. The inflammatory response detailed in this study appears to be unique to elastase and cannot be reproduced with other serine proteinases, such as trypsin and chymotrypsin, or a cysteine proteinase, papain. Our data has also shown that the proteolytic activity of elastase can be inhibited by an elastase-specific inhibitor, elastatinal, and by doing so attenuates the TNFα response; elastase stimulation alone produced 127.5±72.1pg/mg tissue, whereas with the inhibitor this production dropped to 40.4±9.0pg/mg tissue. As of yet, the exact mechanism by which elastase induces inflammation is unknown but we have investigated the relationship between elastase and two candidate receptors-proteinase-activated receptor (PAR)-2 and Toll-like receptor (TLR)-4. Although elastase stimulation does not appear to alter the gross amount of these receptors present in the parenchyma, we have found that those patients with mild to moderate COPD tend to have greater levels of both PAR-2 and TLR-4. We have also utilised synthetic activating peptides for PAR-2 and in comparison to elastase stimulation it is suggested that elastase may cause its inflammatory and Th2 effects via distinct pathways.

Rhinovirus (RV) infection is a major cause of asthma exacerbation in children and adults. Previous studies have shown that primary bronchial epithelial cells (PBECs) obtained from asthmatic subjects have a deficient interferon (IFN) response against RV infection, the molecular mechanism of which is unknown (Wark et al 2005; Contoli et al 2006). Initially it was hypothesised that this deficiency is inherent to other cell types in the airway, such as bronchial fibroblasts. In a rhinovirus infection model, it was shown that fibroblasts respond with a vigorous pro-inflammatory response. However this response was accompanied by no significant IFN response. Fibroblasts produced IFN when we treated with a synthetic double-stranded RNA. However, no differences were observed between normal and asthmatic cells, indicating that the deficient innate immune response in asthmatic epithelium is not inherent to other cell types. We suggest that in vivo, bronchial fibroblasts may contribute to the inflammatory state in asthma when infected with RV. This might occur when an epithelial cell barrier with disrupted tight junctions might allow penetration and infection of the underlying mesenchymal cell layer. To investigate the innate immune response of asthmatic PBECs we hypothesised that the anti-inflammatory cytokine transforming growth factor beta (TGF-β) dampened the innate immune response against rhinovirus infection. It has been shown previously that TGF-β is elevated in asthmatics. It was found that PBEC cultures from asthmatic subjects produce significantly more endogenous TGF-β2 compared to healthy controls. When PBECs from healthy donors were treated with exogenous TGF-β2 it promoted RV replication, which was coupled with a decreased IFN response. Conversely, treatment of PBECs from asthmatic subjects with a neutralizing antibody against TGF-β decreased RV replication. These observations provide an interesting link between an anti-inflammatory environment in the asthmatic airways contributing to a defective innate immune response in asthma. To understand the TGF-β-mediated effect on RV replication, the importance of src kinases as one of the upstream signaling molecules in TGF-β-dependent alterations of cellular physiology was investigated. We found that inhibitors of src, in particular the SU6656 compound, were very potent in inhibiting RV replication. Inhibition by SU6656 was coupled with a significant increase in IFN response. These findings may pave the way towards designing compounds of similar structure, which are able to augment the IFN response and therefore provide a new form of therapy against asthma exacerbation.

Asthma is a heterogeneous condition resulting from a complex interaction of genetic and environmental factors leading to airway hyperreactivity, reversible bronchoconstriction and chronic airway inflammation. Little is known about the role of chronic bacterial colonization in the underlying inflammation and how it can alter disease phenotype. In COPD, airway colonization with potentially pathogenic microorganisms (PPMs) leads to neutrophilic airways inflammation, increased frequency of exacerbation, worsening symptom scores and an accelerated decline in lung function. The Gram positive coccus Staphylococcus aureus is known to produce enterotoxins (SAEs) which can act as superantigens leading to an exaggerated T lymphocyte response and has been linked to steroid resistance in asthma. Hypotheses tested were: (1) Bacterial colonization patterns differ between the asthmatic and healthy lower airway. (2) Airway colonization with potentially pathogenic bacterial species contributes to asthma severity and alters phenotype. (3) S. aureus colonizes the lower airway in asthma and contributes to treatment resistance through the action of superantigens. The non1culture based, molecular microbiological technique of terminal restriction fragment length polymorphism profiling (T1RFLP) was used to demonstrate the patterns of airway bacterial colonization in severe asthmatics, mild asthmatics and healthy controls. We have demonstrated that T1RFLP profiling of respiratory specimens is a more sensitive tool than standard respiratory culture in detecting potentially pathogenic bacterial species and is able to give a relative abundance of each species compared to the total bacterial load within the specimen. Bronchoalveolar lavage (BAL) was used to sample the distal lower airways of volunteers and demonstrated a more diverse colonizing flora in severe asthmatics than mild asthmatics (p=0.001) and healthy controls (p=0.029). PPMs such as Haemophilus sp. and Streptococcal sp. were more frequently detected in severe asthma than in mild asthma (p<0.001) and healthy controls (p=0.004). The total abundance of PPMs as a percentage of total bacterial load was significantly higher in severe asthmatic BAL than mild asthmatic (p<0.001) and healthy subjects (p=0.004). Within the severe asthma group total BAL PPM abundance correlated with neutrophil counts, IL18 and worsening FEV1. Induced sputum was used to sample the central, proximal, lower airways and demonstrated lower species diversity in severe asthma than in mild asthma (p=0.012) and healthy controls (p=0.005). Within the severe asthma group, when the most abundant species within induced sputum was Haemophilus sp., Streptococcal sp. or Moraxella catarrhalis, significantly worse FEV1 (p=0.025), higher sputum neutrophil counts (p=0.001) and longer disease duration were seen. Dominance of these species within induced sputum was associated with a neutrophilic asthma phenotype (p=0.014). Evidence of the effect of S. aureus superantigens was investigated with BAL T cell receptor (TCR) V? profiling. BAL S. aureus colonization was demonstrated in 38% of severe asthmatic subjects and was associated with higher V? 5.3 (p=0.001), V? 5.1 p=0.01), V? 13.1 (p<0.001) and V? 2 (p=0.022) positive CD4 T cells. This skewing of TCR V? populations provides a surrogate marker for the action of SAEs which may be relevant in the development of steroid resistant disease. The findings of clinically relevant PPM airway colonization in severe asthma provide an opportunity for new targeted therapies in a group of patients where there are currently few therapeutic options.

Cystic Fibrosis (CF) causes chronic lower respiratory tract infection leading to morbidity and mortality. CF Pulmonary Exacerbations (CFPEs) cause accentuated symptoms and increase mortality. The definition and aetiology of CFPEs has however proved elusive. Recently, culture independent techniques have shown that there is much greater diversity of bacteria than previously detected by culture dependent methods. Building on this, in the work presented here, the bacteria in respiratory samples from adults with CF were studied over a 12 month period. Each subject provided thrice weekly sputum samples for analysis by culture and culture independent microbiological methods. Concurrently an in-depth assessment of their subjective and objective health using Visual Analogue Scales (VAS) and spirometry (FEV1) was undertaken. Inflammation markers were also measured. A total of 2061 samples from fourteen adults (mean age 30.2; mean FEV1% predicted 53.3%; 6 females; 8 ?F508 homozygotes) were collected. Subjective VAS measures correlated with objective spirometric measures. However, previously unsuspected complexity of subjective symptomatology was found. Ribosomal clone sequence analysis identified 90 different species, including 15 not previously reported in CF lung disease. Notably 44% of species detected were obligate anaerobes, and 72% were species previously associated with the human oro-pharynx. During the study period, subjects experienced 42 CFPEs requiring treatment. New species were not seen to enter the bacterial community as aetiological agents for CFPEs. However, whilst treatment for CFPEs caused a large fall in the proportion of anaerobic species, no significant change in the proportion of Pseudomonas aeruginosa was detected. Significant and potentially important differences in bacterial community composition, structure and stability between subjects separated by gender, genotype and lung function were observed. Moreover, the presence of certain species correlated with subjects suffering frequent CFPEs. The results presented here give new insights in to the complexity of symptoms and bacterial diversity in CF pulmonary disease

The origins of respiratory disease, such as asthma in childhood and COPD in later life are unclear. Maternal smoking during pregnancy and low birth weight is associated with increased risk of asthma, poor lung function in adults and COPD in old age. Exposure to oxidative stress and poor nutrition in utero is thought to cause damage to the lung and alter the normal course of lung development. Glutathione S-transferases (GST) are potent antioxidants. In this work, genetic polymorphisms that alter GST enzyme activity were genotyped in a family-based childhood asthma cohort (341 families, n = 1508) and analysed to investigate whether they alter the risk of developing asthma when individuals are exposed to environmental tobacco smoke. Real-time PCR based copy number variation methodology was developed to genotype the common gene deletion polymorphism of GSTT1 and GSTM1 genes, for other GST genes (GSTP1 and GSTO2) SNP haplotypes were constructed. A rare GSTO2 haplotype was negatively associated with asthma susceptibility, atopy severity, and FEV1 values. Asthmatic children with a GSTT1 gene deletion, or a common GSTP1 haplotype, developed more severe asthma compared to individuals with a GSTT1 gene or non-carriers of the GSTP1 haplotype. Total IgE levels were increased in GSTT1*0 individuals when exposed to tobacco smoke in early life, suggesting a gene-environment interaction. GSTO2 may be a shared susceptibility locus for asthma in childhood and COPD in later life. Animal models of maternal protein-restriction during pregnancy can induce hypertension, diabetes and endothelial dysfunction in offspring and in some of these models alterations to lung gene expression and lung architecture have been reported. This work established that a rat model of maternal dietary protein-restriction during pregnancy known to induce hypertension in the offspring, results in persistent alterations to the expression of genes in the lungs of adult offspring (120 days), including genes involved in glucocorticoid action (Hsd11b2), growth (Igf1 & 2 and Pcdh1) and alveolar development (Tp53). Lung microRNA expression profiles were also altered in response to exposure to protein restriction in utero. These findings suggest a role for nutritional programming in respiratory disease susceptibility in later life and a role for microRNAs in the study of the developmental origins of health and disease in general. Further work will include the investigation of epigenetic mechanisms that control nutritional programming in lungs of animals exposed to protein-restriction in utero. This work has demonstrated that GST polymorphism is a risk factor for childhood asthma and certain genotypes can offer some protection against the development of severe asthma. There was little evidence to suggest that GST polymorphism modulates the effects of smoke exposure in early life. In addition, we have demonstrated that maternal diets that are poor in nutrition could predispose her offspring to respiratory disease in later life by altering the course of normal lung development in early life or response to environmental stimuli in later life.

COPD (Chronic Obstructive Pulmonary Disease) worldwide has a prevalence of 10% in men and 8.5% in women. Exacerbations of COPD account for approximately 10% of all acute medical admissions. Projected prevalence figures suggest that by 2020 COPD will be the third leading cause of mortality worldwide thus imposing a significant burden on healthcare resources in the future. Acute exacerbations are not only responsible for a decline in the patient’s quality of life, but have a major socioeconomic impact. Following a pilot study that showed current smokers recover lung function much more slowly from their exacerbation than ex smokers, I initiated a properly powered prospective study to investigate the difference between the two groups. A total of 58 patients admitted with acute infectious exacerbations of COPD were recruited to the study to determine the effect of smoking status on their exacerbation. Throughout the admission lung function was measured. Sputum was cultured for bacteria, and PCR used to detect viral infection. Blood and sputum cells were analyzed by flow cytometry. Serum was collected for CRP levels. Ex-smokers recovered significantly more quickly than current smokers in all spirometric parameters (P<0.01), and were discharged sooner (mean 3.08 vs 5.59 days, P<0.001). Sputum culture was positive for more pathogenic bacteria in current smokers, especially H. influenzae, which was associated with a significantly higher CRP rise (p<0.05) than any other organism. CD8+ T cells predominated in the sputum of ex-smokers while CD4+ T cells were the dominant cell type in current smokers (p<0.01). Current smoking is a risk factor for more severe exacerbations, delayed recovery and prolonged hospitalization. This may result from a variety of factors including bacterial, immune mediated responses and systemic inflammation.