Background Activation of the innate immune system plays a key role in exacerbations of chronic lung disease, yet the role of fibroblasts in innate immune responses and the identity of danger signals (alarmins) that may contribute to their activation are still to be unraveled. The objectives of this study were to identify epithelial alarmins released during environmental insults which induce innate immune responses in lung fibroblasts and dissect the mechanisms responsible for this. Methods Primary human lung fibroblasts (PHLF) were treated with conditioned media from primary bronchial epithelial cells (PBECs) exposed to oxidant injury or endoplasmic reticulum stress (ER stress) and fibroblast responses assessed. The relevance of alarmins in-vivo was assessed clinically by measurement of relevant alarmins longitudinally in patients developing Bronchiolitis Obliterans Syndrome (BOS) after lung transplantation. Results Conditioned media from PBEC cells subjected to oxidant injury and ER stress contained elevated levels of alarmins. Treatment of PHLFs with conditioned media from damaged cells significantly upregulated IL-6, IL-8, MCP-1, GM-CSF, IL-1α and IL-1β expression (p<0.05). This effect was reduced with anti-IL-1α or IL-1Ra but not anti-IL-1β antibody. Co-stimulation with Poly I:C significantly accentuated the IL-1α induced inflammatory phenotype in PHLFs. Clinically, IL-1α was increased in BAL of lung transplant recipients with infections and within 3 months of developing bronchiolitis obliterans syndrome (BOS) (p<0.001). Additionally, IL-1α levels positively correlated with elevated IL-8 (p<0.001) and neutrophil counts (p<0.001). Conclusions IL-1α plays a pivotal role in triggering proinflammatory responses in fibroblasts and this process is accentuated in the presence of viral pathogen associated molecular patterns. This novel pathway warrants further evaluation of its therapeutic potential to limit the repeated cycles of injury and exacerbation in chronic lung diseases.

Cancer is a leading cause of mortality throughout the world and new treatments are urgently needed. Recent studies suggest that bone marrow-derived mesenchymal stem cells (MSCs) home to and incorporate within tumour tissue. This property can be utilised to deliver targeted anticancer therapies. This thesis describes the production of MSCs engineered to express TNF-related apoptosis-inducing ligand (TRAIL), a transmembrane protein that causes selective apoptosis of tumour cells. Human MSCs were transduced with TRAIL and the IRES-GFP reporter gene using a lentiviral vector, under the control of a tetracycline promoter. Transduced and activated MSCs caused lung, breast, squamous, and cervical cancer cell apoptosis in vitro. In vivo, the cells were able to specifically home to tumours and both significantly reduce tumour growth, and eliminate metastatic disease. The data included in this thesis demonstrates for the first time a significant reduction in metastatic tumour burden with frequent eradication of metastases using inducible TRAIL-expressing MSCs. This has a wide potential therapeutic role, which includes the treatment of both primary tumours and their metastases, possibly as an adjuvant therapy in clearing micrometastatic disease following primary tumour resection.

Patients with refractory asthma represent a significant unmet clinical need where treatment options are limited and effective management poses a challenge. Treatment with daily systemic corticosteroids is the norm for many patients. Subsequently they are at risk of many significant and potentially life threatening adverse effects of systemic corticosteroid use. Little evidence is available surrounding this issue in a severe or refractory asthma population. Using data from the British Thoracic Society Difficult Asthma Registry and the Optimum Patient Care Research Database, the aims of this thesis' were to determine the prevalence of systemic corticosteroid dependency in refractory asthma and the direct healthcare costs associated with this cohort and finally to identify the prevalence I of systemic corticosteroid induced morbidity in refractory asthma. We identified that ca 52% of patients with refractory asthma in the UK are receiving daily doses of systemic corticosteroids. We found that the economic costs of patients with severe refractory asthma attending specialist difficult asthma centres are substantial and outweigh the costs of patients with difficult to treat asthma. Furthermore, we determined the cross-sectional prevalence rates for adverse effects which are commonly related to systemic corticosteroid use in severe refractory asthma including type 11 diabetes, osteoporosis, gastric disorders, obesity, hypertension and obstructive sleep apnoea. These are Significantly greater in subjects with severe asthma who require daily systemic corticosteroids, compared to subjects with severe disease using intermittent rescue corticosteroids and well-controlled asthma In conclusion, we have identified that refractory asthma represents a substantial burden to the NHS as well as being associated with many adverse effects related to systemic corticosteroid exposure. The need for better targeting of steroid therapy in asthma using objective biomarker based strategies and the urgent delivery of novel therapies which will both reduce exposure to systemic corticosteroids and the overall burden of disease are critical

Tobramycin, an aminoglycoside, is one of the mainstay treatments for chronic infections of pseudomonas Aeruginosa within the CF lung to decrease the bacterial burden. In in vivo studies tobramycin is highly potent against P. Aeruginosa, however, when administered to the CF lung large dosages are required to reach near MIC concentrations due to poor penetration of the CF mucus. These high dosages required lead to unwanted side effects and with the poor penetration of the CF mucus, the full therapeutic potential of tobramycin is unfulfilled. The focus has therefore been around the development of methodologies to functionalise tobramycin in order to facilitate the enhanced delivery to the CF lung using either covalent of non-covalent modification. One issue of tobramycin is its poor solubility in the majority of molecular solvents; as such the solubility of tobramycin was investigated within a series of hydrophilic ionic liquids. The solubility study proved successful, in which it was demonstrated that hydrophilic ionic liquids can solubilise tobramycin to a high degree, far surpassing that of conventional molecular solvents. Attempts to functionalise tobramycin using either P(III) or P(IV) reagents proved troublesome with little success, however, selective modification of the primary amine of tobramycin was achieved through potentially labile carbamate linkers. Attempts to conjugate these analogues of tobramycin with a functionalised analogue of SLPI, devised to act as a delivery vector was unfortunately not successful

Aim: To investigate the clinical, physiological, radiological and pathological changes which occur in the lungs in Anderson Fabry Disease (AFD) Methods: In this study we have used lung function testing, high resolution CT scanning and induced sputum examination to investigate the lung. We have measured sputum enzyme activity using fluorometric assays, cell populations using flow cytometry and cytokines using enzyme linked immunosorbent assays. We have compared investigation findings from AFD subjects with those from patient’s with airways disease in the form of chronic obstructive pulmonary disease (COPD) and healthy controls Results: We have shown that respiratory symptoms are common, and airway involvement is widespread though mild in AFD. Pulmonary involvement is more common in males, in subjects with worse overall disease as measured by Mainz Severity Score Index, and is independent of smoking. No significant radiological changes were evident on CT chest imaging in AFD. We have presented novel data on α-galactosidase A activity from lung derived samples, which demonstrate low sputum enzyme activity in AFD males compared to controls and AFD females, and consistently higher enzyme activity in sputum derived leucocytes compared to those derived from peripheral blood. We did not find any detectable differences in blood or sputum α-galactosidase A activity in subjects on enzyme replacement therapy. Cell populations from induced sputum in AFD subjects demonstrated a predominance of monocytes/macrophages, similar to the COPD subjects, and there was the suggestion of an increased T cell population in AFD subjects with airway obstruction compared to those without. Elevated concentrations of sputum IL-8 were seen in the sputum of AFD subjects compared to controls. Conclusion: There is demonstrable and clinically relevant involvement of the lungs in AFD, which appears to occur as a result of deficient α-galactosidase A in the lungs and subsequent inflammatory processes.

Non-invasive respiratory support (NIV) is associated with a high prevalence of local side- effects which may be associated with induction of upper airway inflammation. This thesis examines the effect of NIV on the upper and lower airway by examining bronchial epithelial cell cultures, healthy subjects, obstructive sleep apnoea (OSA), and chronic obstructive pulmonary disease (COPD) patients. The rationale for this thesis is based on reports suggesting that continuous positive airway pressure (CPAP) may induce early upper airway inflammation, and discusses the relationship between upper and lower airways in COPD. To examine the addressed rationale we used both an in vitro and in vivo approach. In vitro, we examined the release of the inflammatory cytokines from a human bronchial epithelial cell line over time-intervals using CPAP therapy. In vivo studies investigated whether induction of nasal inflammation was associated with the development of systemic inflammation, nasal symptoms, and changes in nasal mucociliary clearance after a short period of CPAP therapy. Results were investigated further in OSA by investigating whether induction of nasal inflammation was associated with the development of systemic inflammation, nasal symptoms, airway obstruction, and impaired adherence to CPAP therapy and quality of sleepiness over a six months follow-up period. Additional pilot data obtained involved a comparison of local and systemic inflammatory indices in COPD patients using and not using NIV. The key finding was that CPAP is pro-inflammatory. The in vitro data showed that CPAP resulted in the release of inflammatory mediators from cultured human bronchial epithelial cells, in a time-and-pressure-dependent manner. Meanwhile, in vivo data from healthy control subjects showed CPAP was associated with dose (pressure) response changes in nasal and systemic inflammatory markers, reduced nasal function, and the development of nasal symptoms. The development of nasal symptoms was associated with the degree of functional impairment and nasal inflammatory response. These in vitro and in vivo results were novel in reporting the effects of CPAP in this way, providing new data on the mechanisms of CPAP intolerance in the crucial, early phase of therapy. Furthermore, the long-term CPAP study with OSA resulted in nasal inflammation, reduction in nasal mucociliary function, and significant other adverse effects. However, sleep quality and the perceived benefits of therapy improved over time, despite the presence of side-effects. These results have important implications for clinical practice, since it demonstrates a relationship between nasal symptoms, mucociliary clearance, inflammation and compliance in patients with OSA initiating and continuing CPAP therapy. Further investigation of strategies to combat the initial side-effects and nasal inflammation associated with this treatment modality might target the epithelial lining of the nose in an attempt to address the origin of the inflammatory response. In addition, educational and support strategies to improve patients‟ tolerance of side effects may further increase compliance with nasal CPAP treatment for OSA patients.

3q amplification is the most common genomic aberration in squamous lung cancer, suggesting that a ‘driver’ oncogene for squamous lung cancer resides within the 3q amplicon. Studying preinvasive disease has the theoretical advantage of identifying the key early molecular events in the pathogenesis of squamous lung cancer. This thesis was based on developing a method (microdissection-Molecular Copy-number Counting - μMCC) for the analysis of regional genome structure in archived biopsies and then applying this method to the analysis of the 3q amplicon in a rare collection of preinvasive bronchial lesions. Molecular Copy-number Counting was adapted and validated for the assessment of copynumber variation in degraded DNA extracted from microdissected formalin-fixed paraffin-embedded biopsies. This approach should be generally applicable, both in basic cancer research, and in the stratification of treatment based on the analysis of genome structure in clinical biopsies. A further series of experiments on high molecular weight DNA from peripheral blood raised unexpected technical issues about the effect of DNA integrity on single molecule PCR; evidence is provided suggesting that DNA sequences with very high melting temperature external to the target sequence may inhibit PCR efficiency. μMCC was applied to a series of low- and high-grade dysplastic lesions. There were a number of novel findings. Firstly, genome evolution is a dynamic process and corresponds to histological grade. All high-grade, but no low-grade lesions, showed gain or amplification of 3q relative to 3p. No low-grade lesions progressed to cancer. Secondly, the minimal commonly amplified region was narrowed to a 3.5Mb region encompassing 17 genes with the two outstanding candidates being PIK3CA and SOX2. Finally, for three individual patients, the analysis of series of biopsies sampled longitudinally allowed new observations on clonal expansion and suggested incremental regional amplification may be important in the progression of preinvasive lesions to invasive cancer.

Rationale It had been reported that alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) display glucocorticoid resistance. Previous studies have only investigated limited numbers of inflammatory mediators. Objectives To compare the Gc sensitivity of a range of inflammatory mediators produced by LPS stimulated alveolar macrophages from capo patients compared to smokers (S) and healthy non-smokers (HNS). Methods and measurements LPS stimulated alveolar macrophages from 15 capo patients, 9 Sand 9 HNS were stimulated with LPS with or without dexamethasone (100 and 1000 nM). Luminex and ELISA were used to measure 23 inflammatory mediators. Combination treatment with dexamethasone and p38 MAP K inhibitors was also analysed. Main Results After LPS stimulation there were lower levels of inflammatory mediators in COPD patients and S compared to HNS, reaching statistical significance (p<0.05) for GROa, IL-8 and GM-CSF. Neither dexamethasone concentration inhibited GM-CSF or G-CSF production in any of the 3 groups. There was significant (p<0.05) inhibition ofGRO-a, IL-8, MIPla, IL-6, TNF-a, IL-lO, Rantes and MCP-l production in all 3 subject groups. There was no difference between groups for the effects of dexamethasone at either concentration (p>0.05 for all comparisons). TNF-a, IL-6 and GRO-a displayed the most sensitivity to dexamethasone in CO PO patients, while IL-8, GMCSF and GCSF were the least sensitive. Treatment with p38 MAP K inhibitors maximised inhibition of Gc insensitive mediators. Conclusions COPD macrophages have a reduced response to LPS. Glucocorticoid sensitivity was similar in capo macrophages compared to controls. Some glucocorticoid insensitive cytokines were identified, including GM-CSF, G-CSF and lL-8 that may be involved in the progression of airway inflammation in patients treated with glucocorticoids. Inhibition was maximised using combination treatment with p38 MAPK inhibitors.

Rationale Cough is an important symptom in asthma. In addition, cough is both troublesome to patients and may be important in the development of more severe disease. Moreover, it is a key symptom in the diagnosis of asthma, according to international guidelines. Little is known regarding the mechanism of cough in asthma. Until now, subjective measures of cough have been used in mechanistic and therapeutic studies relating to both cough in asthma and chronic cough. There is no published literature documenting cough rates in asthma or the relationship between cough rates and both subjective measures of cough and asthma control. In addition, the relationship between objective cough rates and airway inflammation, and the effects of cough on airway function and inflammation is unknown. Methods A series of studies was designed and carried out to answer the above questions. Firstly, a cross sectional study examining the relationships between objective and subjective measures of cough in classical asthma (CA), secondly a longitudinal study examining the relationship between cough rates and CA control and thirdly a cross-sectional study examining the effects of voluntary cough on airway function and inflammation. Results There was a poor correlation between objective and subjective measures of cough in CA. Cough-related quality of life correlated most closely with cough rates. Traditional measures of CA (e.g. spirometry) poorly reflected cough rates. Objective cough rates and sputum eosinophils were predictive of CA control as defined by ACO. Objective cough rates did not reflect sputum inflammatory cell count or mediators. Voluntary cough induced small changes in peak flow in mildmoderate CA and small changes in sputum ECP that were not reflected by changes in sputum eosinophils. Conclusions Subjective measures of cough in asthma are poor substitutes for objective cough rates. When designing studies for cough in asthma, both objective cough rates and cough-related quality of life should be incorporated. Objective cough rates reflect asthma control (independent of airway inflammation) more closely than traditional measures of asthma. Airway inflammation does not appear to directly reflect cough rates. In addition, short bouts of coughing in mildmoderate asthma induce neither significant changes in airway function nor airway inflammation.

No description available.