Lysophosphatidic acid (LPA) is a major serum phospholipid and bioactive lipid mediator that exerts a broad range of effects through its family of cognate G protein-coupled receptors via coupling to multiple G proteins and can affect a variety of cellular functions in a range of tissues and cell-types. LPA is known to affect airway function and a growing body of evidence suggests a role for LPA receptor function in the molecular aetiology of asthma. The aims of this project were to characterise the function of LPA and its receptors in cultured human airway smooth muscle (hASM) cells from asthmatic and control donors. Expression of a broad range of genes related to LPA metabolism/signalling and inflammatory signalling were analysed using both traditional and high throughput RT-qPCR methods. Decreased expression of the genes for a phosphodiesterase, PDE4B, and an LPA metabolising enzyme, LPP2, was detected in hASM cells derived from asthmatic patients compared to control donors. Investigation of LPA receptor pharmacology using a [³⁵S]GTPγS binding assay in a model cell-line showed that LPA species with a range of fatty acid tail lengths and degrees of saturation were active at LPA receptors with varying potencies. LPA exhibited complex modulation of forskolin and isoprenaline induced cAMP responses in hASM cells. Though no differences in the effects of LPA treatment were observed in the two disease-states studied, hASM cells from asthmatics exhibited a significantly higher magnitude of cAMP response compared with those from control donors, which could indicate dysfunctional adenylyl cyclase or PDE activity in disease. These findings confirm that LPA is able to affect hASM cell function in ways pertinent to asthma's pathophysiology, but do not indicate altered LPA receptor expression or function in disease.

Rationale: Airway smooth muscle dysfunction is a cardinal feature of asthma that is believed to contribute to the symptoms and recurrent exacerbations. However, the mechanisms driving this dysfunction are not fully understood. Hypothesis: Intrinsic abnormalities of the ASM from asthma exist and contribute to the functional differences in vitro which correlate with clinical characteristics Methods: Primary airway smooth muscle cells from healthy control subjects and asthmatics were used. Baseline protein and gene expression studies were assessed by mass spectrometry and microarrays. Functional differences were assessed, such as survival, proliferation, migration, wound healing and contraction. Oxidative stress DNA damage was assessed by immunohistology and comet assay, reactive oxygen species (ROS) quantification, and the role of NADPH oxidase (NOX4) and superoxide dismutase (SOD2) were assessed by quantitative gene expression and pharmacological inhibitors, mimetic and gene silencing. Results: In ex vivo airway smooth muscle cultures, asthmatic smooth muscle dysfunction persisted, indicating exaggerated contractility and pro-inflammatory mediator release compared to ASM from healthy controls. The oxidative burden and damage in asthmatic ASM was increased with increased expression of NOX4 which negatively correlated to the airflow obstruction. Agonist induced hyper-contractile response was abrogated by using pharmacological inhibition or by silencing the mRNA. Summary: Intrinsic abnormalities of the airway smooth muscle exist in asthma. Asthmatic ASM cells possess intrinsic abnormalities in gene and protein expression, synthetic mediator production and exaggerated agonist induced contraction. Increased expression of NOX4 in asthmatic ASM promotes the susceptibility of a hyper-contractile response, implicating NOX4 as a potential therapeutic target for the treatment of asthma.

Exacerbations are key events in the natural history of chronic obstructive pulmonary disease (COPD), with limited recovery of physical performance, and the highest cause of readmission in the UK. This thesis explores the impact of exacerbations in COPD and chronic respiratory disease. In the first study I have investigated the effects of an early rehabilitation intervention on healthcare utilisation, strength and exercise capacity by conducting a large randomised control trial. Using a sub-group of this cohort I have then explored factors that predict hospital readmission. Finally I have conducted a study of single leg neuromuscular electrical stimulation (NMES) in stable COPD, alongside a resistance training group. No difference was seen following early rehabilitation in hospitalisation, healthcare utilisation or physical performance. A number of unexpected findings were noted, including an increase in 12 month mortality in the intervention group and large functional recovery in the usual care group. Using multivariate analysis three risk factors for hospital readmission were identified, including quadriceps cross sectional area, using ultrasound. In the stable state NMES was seen to significantly increase muscle mass from baseline, comparable to changes seen using resistance training. In summary early rehabilitation in chronic respiratory disease does not impact on future hospitalisation. Identification of those with rehabilitation potential is required as the hospitalised population represent a frail group, with advanced disease.

This thesis is composed of four clinical trials which examine diagnostic, prognostic and management aspects of pleural malignancy and pleural infection, all contributing novel data to the existing body of knowledge in these disease areas. The studies presented are as follows: • The South West Area Mesothelioma and Pemetrexed (SWAMP) Trial - a prospective observational trial examining markers of prognosis and chemotherapy response in malignant pleural mesothelioma. • A study of the association between plasma and pleural fluid Vascular Endothelial Growth Factor (VEGF) and its soluble receptor (sVEGFR-1) and survival and pleurodesis outcomes in a consecutive series of patients with a malignant pleural effusion . • A prospective observational study of serum and pleural fluid mesothelin testing in the investigation of undiagnosed pleural effusions. • Pleural irrigation trial (PIT): A randomised controlled pilot study of pleural irrigation with normal saline versus standard care in patients with pleural infection.

No description available.

Matrix metalloproteinases (MMPs) have been implicated in the lung destruction seen in lymphangioleiomyomatosis (LAM) as elevated MMP levels have been detected in tissue, serum and urine from LAM patients but a causal link has not been established. It has also been hypothesised that inhibition of MMPs with the tetracycline antibiotic doxycycline may provide a potential treatment for LAM. Previously proposed mechanisms of actions of doxycycline were examined. In Eker rat derived ELT3 cells doxycycline at doses ~25 micrograms/ml inhibited cell proliferation and adhesion, possibly by a toxic effect. This was also seen in human angiomyolipoma-derived cells. There was no effect of doxycycline on MMP-2 expression or activity in vitro. In a xenograft model, doxycycline had no effect on tumour growth, final tumour weight, or tumour lysate MMP levels. The role of MMPs as potential biomarkers was also compared with vascular endothelial growth factor D (VEGF-D). Serum VEGF-D, Angiotensin coverting enzyme (ACE) and total matrix metalloproteinase 2 (MMP-2) levels were elevated in patients. VEGF-D was the strongest discriminator between patients and controls and the addition VEGF-D measurement to ERS criteria reduced the need for biopsy to confirm diagnosis. Finally, we performed a 2 year, randomised, double blind placebo controlled study of doxycycline in 23 patients with LAM and found no evidence that it prevented decline in lung function, or improved exercise tolerance of quality of life.

No description available.

Acute Respiratory Distress Syndrome (ARDS) is characterised by dysregulated inflammation within the lungs, which can severely compromise pulmonary architecture and function. Despite intensive efforts to develop therapies for ARDS, there are no curative pharmacological interventions at present. Instead, management of ARDS is primarily supportive and the syndrome is associated with substantial mortality, morbidity and healthcare expenditure. In pursuit of an effective ARDS therapy, this thesis explored the potential application of a novel sialic acid-functionalised nanoparticle (SNP). This nanoplatform was designed to actively target sialic acid-binding immunoglobulin-like lectin (Siglec) receptors expressed on monocytes and macrophages, which exert a fundamental role in the negative regulation of inflammatory responses. Initially, SNP were evaluated in a murine model of lipopolysaccharide (LPS)-induced lung injury, where promising outcomes were observed. SNP attenuated several lung injury parameters in this model, including neutrophilia and pro-inflammatory cytokine levels in the pronchoalveolar lavage fluid (BALF), amongst others. Crucially, the translational efficacy of SNP was confirmed in human models of LPS-induced inflammation. SNP inhibited pro-inflammatory cytokine production in cultures of primary human cells implicated in ARDS, whilst concurrently enhancing levels of antiinflammatory interleukin (IL)-10. The protective effects of SNP were also evident in whole human lungs injured with LPS ex vivo, where reductions in pulmonary oedema and BALF cellularity were observed. Collectively, these findings highlight the potential of SNP to combat the inflammatory component of ARDS and fulfill the clinical need for an effective therapy.

Bronchiectasis is a common clinical problem with great variance in its aetiology. treatment and prognosis. Lung function assessment using spirometry, and in particular FEV1 is used to objectively assess the degree of airflow impairment present. This however has limitations in its use, as it is frequently normal in mild to moderate disease and in many clinical trials does not show evidence of responsiveness to interventions. The lung clearance index (LCI), derived from multiple breath washout (MBW) testing has been shown in CF to have greater sensitivity in assessing lung function by determining ventilation inhomogeneity than indices of airflow obstruction. The aims of this thesis were to assess the reliability, validity and sensitivity of Lel as a marker of lung disease in bronchiectasis. This project has shown that LCI has good intervisit repeatability and is a feasible test for use in the clinical setting assessing 30 patients who are clinically stable on 2 occasions. 2 weeks apart. LCI has also been shown to have superior sensitivity to FEV1 when comparing radiological changes using the gold standard of high resolution eT scanning in 60 patients at a single visit when Clinically stable. However, pilot data in a responsiveness study in 9 patients receiving treatment for an infective exacerbation using intravenous antibiotics showed no statistically significant changes in LCI as a marker of responsiveness. This requires a larger. multicentre driven approach to assess the true responsiveness of LCI and other treatment interventions. Overall these results show that LCI by MBW is a reliable, sensitive and feasible measure of lung function in bronchiectasis. The results and the lessons learned in this area can guide future studies to use LCI as a key outcome measure in the management of bronchiectasis both clinically and in the research environment.

Background. Respiratory syncytial virus (RSV) causes recurrent epidemics in communities and repeated reinfections of individuals throughout life. The extent to which this is a consequence of antigenic diversity of the virus caused by genetic evolution is poorly defined. Clarification of these phenomena has implications for our understanding of RSV persistence and vaccine control. Further, studying RSV epidemics genetic composition contributes to understanding of the transmission dynamics of this virus. Methods. RSV positive specimens were available from the freezer archives of three epiderrnologic studies in KVifi District, Coastal Kenya, undertaken between 2002 and 2012. Selected specimens were nucleotide sequenced in the highly variable attachment (G) protein gene (n=917) or for whole genomes (n=83), and phylogenetically analyzed. This analysis was supported by a set of contemporaneous sequences from around the world deposited in GenBank. Results. The genetic relatedness of virus sequences from infection-reinfection pairs from individuals followed within a birth c.ohort (2.002-05) revealed that the vast majoritY (>90%, 48/53) differed by either RSV group, genotype or G amino acid sequence. However, the genotype temporal distribution amongst re-infections mirrored the contemporaneous distribution in the wider study population.