Computer based CT analysis of interstitial lung disease (ILD) may finally have come of age with sophisticated new software algorithms that can analyse CTs of patients with idiopathic pulmonary fibrosis (IPF) with similar, or superior, precision to visual scoring by radiologists. However, the latest iterations of such tools have not been evaluated in large populations or in fibrosing lung diseases (FLD) other than IPF. The investigations in this thesis aimed to identify the role of a computer-based tool CALIPER, compared to visual CT scoring and pulmonary function indices, in predicting prognosis at baseline in a large IPF cohort and across a range of FLDs. A second line of enquiry was exploration of the utility of serial CT as a prognosticator in IPF patients. Visual scores for these investigations were provided by four experienced sub-specialty thoracic radiologists. Baseline CT analysis in IPF demonstrated that CALIPER parameters, particularly the percentage of the lung occupied by vessels (parenchymal vascular percentage - PVP), were strong predictors of pulmonary function indices as well as mortality. CALIPER variables were the strongest predictors of mortality in patients with hypersensitivity pneumonitis and connective tissue disease-related-ILD and were one of the strongest predictors of mortality in an “all-comers” cohort of FLD. Serial evaluation of CT scans demonstrated that change in CALIPER variables, specifically change in PVP, was a stronger and more sensitive predictor of survival than the best available measure of disease worsening in IPF, namely change in forced vital capacity (FVC). It is concluded that CALIPER is a viable complementary tool in the baseline and serial evaluation of IPF and the baseline analysis of the majority of FLDs. Change in PVP, in particular, may represent a new index in the clinical evaluation of disease progression in IPF and could represent a co-endpoint alongside FVC change in clinical trials.

Severe asthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases of the lung both characterised by airway limitations but with differing pathophysiology. Asthma is normally well controlled with the use of corticosteroids (CS). However in 5 – 10% of all patients with asthma - severe asthmatics - and in most patients with COPD, the response to CS is poor and these patients are relatively CS-insensitive. p38 mitogen-activated protein kinase (MAPK) is a kinase cascade whose heightened activity has been reported in severe asthma and COPD. In this thesis I hypothesised that increased p38 MAPK activity contributes to CS-insensitivity in severe asthma and COPD Induced p38 MAPK activity was increased in peripheral blood mononuclear cells (PBMC) from patients with severe asthma compared with non-severe asthma and in patients with COPD compared with smoking subjects. The ability of dexamethasone to suppress induced pro-inflammatory cytokine release was impaired in PBMC from patients with severe asthma compared with those with non-severe asthma, and in patients with COPD compared with smoking subjects, demonstrating CS-insensitivity in these patients. The inhibition of p38 MAPK activity with GW856553 improved the ability of dexamethasone to suppress induced cytokine release in PBMC from patients with severe asthma or COPD. To investigate the molecular mechanisms by which p38 MAPK activity may be involved in CS-insensitivity, the effect of p38 MAPK inhibition on the phosphorylation of glucocorticoid receptor (GR) at serine 211 residue was determined and was revealed to be p38 MAPK dependent in PBMC from patients with COPD. MAPK phosphatase (MKP)-1 is an anti-inflammatory mediator that is induced by CS and can regulate p38 MAPK activity through dephosphorylation of its serine/threonine residues. Baseline and induced expression of MKP-1 was reduced in PBMC and monocytes from patients with severe asthma compared with non-severe asthma. Impairment of MKP-1 induction by CS may therefore be a mechanism through which CS-insensitivity manifests itself in severe asthma, however, siRNA knockdown of MKP-1 in monocytes from normal subjects resulted in an increase of induced pro-inflammatory cytokines, but it did not reach significance. CS and long-acting β2 agonists (LABA) are used in combination to treat patients with COPD or severe asthma. The effect of fluticasone propionate (FP) or the novel CS, fluticasone furoate (FF), alone or in combination, with a novel ‘ultra-LABA’ vilanterol trifenatate, on suppression of induced cytokine release was examined and compared between severe asthmatics and non-severe asthmatics, and between patients with COPD and healthy smokers. FF, compared with FP, was superior in its ability to suppress induced cytokine release in PBMC from all groups. Moreover, suppressibility of FF in combination with vilanterol, on induced cytokine release, was significantly enhanced, compared with FP in combination with vilanterol, or vilanterol alone, in PBMC from severe asthmatics or COPD.

Acute asthma exacerbations cause significant morbidity and healthcare burden. Up to 60% of acute exacerbations are associated respiratory viruses, particularly human rhinoviruses. The role of bacteria in acute exacerbations is unclear yet antibiotics are frequently prescribed. Recent studies revealed a greater abundance of potentially pathogenic bacteria (e.g. Haemophilus spp.) within the airway microbiota in asthma, whilst a greater abundance of commensals (e.g. Prevotella spp.) were observed in health. The current project examined the dynamics of the airway microbiota in the context of a virus-induced asthma exacerbation. The airway microbiota was assessed in a cohort of mild/ moderate asthmatic subjects. Sputum samples were obtained at baseline and following naturally-occurring cold and underwent 16S rRNA gene sequencing. During acute cold, increased relative abundance of Neisseria sp. (Neisseria_2974) significantly correlated with greater peak flow (PEF) decline and IL-1b level. In contrast, Prevotella and Veillonella sp. (Veillonella_10839) relative abundances correlated with reduced PEF decline and lower IL-1b and IL-8 levels respectively. To validate these findings and evaluate the impact of human rhinovirus on the airway microbiota, a cohort of moderate asthmatic and healthy subjects were experimentally infected with rhinovirus-16. Bronchoalveolar lavage was obtained at baseline and at two time-points post infection. The microbiota community between asthmatic and healthy subjects did not differ significantly at baseline or post rhinovirus-16 infection. Following rhinovirus-16 infection, increased Neisseria_2074 relative abundance again correlated with greater PEF decline whilst increased Prevotella relative abundance correlated with reduced clinical symptoms. Furthermore, rhinovirus-16 viral load exhibited a significant linear relationship with the extent of microbiota community change, suggesting that severity of rhinovirus-16 infection may directly impact on the microbiota. In conclusion, an imbalanced airway microbiota was associated with greater PEF decline and pro-inflammatory cytokine levels during a virus-induced asthma exacerbation, though the precise role of the microbiota remains to be determined.

Pulmonary fibrosis is a progressive scarring disorder of the lung with a dismal prognosis and no curative therapy. Clusterin, a multifunctional glycoprotein with extracellular chaperone activity and involved in regulating cell function, is reduced in bronchoalveolar lavage fluid of patients with pulmonary fibrosis. However, its distribution and role in normal and fibrotic lung are incompletely characterised. Immunohistochemical localisation of clusterin in human lung revealed strong staining associated with fibroblasts in control lung and morphologically normal areas of fibrotic lung but weak or undetectable staining in fibroblasts in fibrotic regions and particularly fibroblastic foci. Clusterin also co-localised with elastin in vessel walls and additionally with amorphous elastin deposits in fibrotic lung. Analysis of primary lung fibroblast isolates in vitro confirmed the down-regulation of clusterin expression in fibrotic compared with control lung fibroblasts and further demonstrated that TGF-β1 is capable of down-regulating fibroblast clusterin expression. shRNA-mediated down-regulation of clusterin did not affect TGF-β1-induced fibroblast-myofibroblast differentiation but inhibited fibroblast proliferative responses and sensitised to apoptosis. Together, these data demonstrate that clusterin promotes lung fibroblast proliferation and survival. Down-regulation of clusterin in fibrotic lung fibroblasts at least partly due to increased TGF-β1 may, therefore, represent an appropriate but insufficient response to limit fibroproliferation. Reduced expression of clusterin in the lung may also limit its extracellular chaperoning activity contributing to dysregulated deposition of extracellular matrix proteins. Alveolar macrophages express clusterin receptor LRP2, suggesting that these cells are responsive to altered clusterin in the lung. In vitro studies with human alveolar and blood-derived macrophages, demonstrate that exogenous clusterin induces the secretion of pro-inflammatory cytokines/chemokines, including TNFα, suggesting a clusterin-mediated polarisation towards an “M1-like” phenotype. Reduced levels of secretory clusterin in the fibrotic lung may, therefore, benefit polarisation towards “M2-like” macrophages, which produce pro-fibrotic mediators, including TGF-β1, resulting in further clusterin reduction and progression of pulmonary fibrosis.

The fungal kingdom contains well over a million species, of which about 80,000 have been named and approximately 600 species cause some form of human disease. Fungal spores are ubiquitous in the airborne environment and inhaled daily often in large numbers though seldom lead to disease. Atopy is a common clinical feature of asthma highlighting the genetic and environment interactions that give rise to clinical phenotypes. Allergy towards fungi is recognised to play an important role in asthma and fungal sensitisation in asthma is associated with an increased risk of multiple hospital and ITU admissions. It is thought that fungal allergy arises from a disproportionate Th2 response to fungal allergens present in spores and hyphae. The risk of IgE sensitisation to fungi may be increased by the capacity of some thermotolerant fungi (typified by Aspergillus fumigatus), to colonise the airways, with an extreme though unusual form exemplified by the condition allergic bronchopulmonary aspergillosis (ABPA). This relationship however is imperfectly understood and lesser displays of fungal allergy in severe asthma are increasingly recognised. The aim was to characterise the relationship between fungal colonisation as defined by a positive sputum culture, fungal sensitisation and the clinical features of mainly moderate-severe asthma. Secondly, this information would guide a placebo controlled trial targeting airway colonisation with Voriconazole. Over 25 species of filamentous fungi were cultured from asthmatics sputum with the flora dominated by Aspergillus fumigatus. There was a correlation between IgE sensitisation to A. fumigatus and evidence of lung damage defined by fixed airflow obstruction and bronchiectasis. There was a demonstrable association between lung damage and fungal colonisation. A three-month randomised trial of oral Voriconazole in patients with asthma and Aspergillus sensitisation failed to improve asthma control or reduce exacerbations. The place of Voriconazole in patients with fungal associated asthma remains to be established.

Spinal cord injury (SCI) disrupts the communication between the brain and the spinal cord. Rehabilitation methods have focused on promoting activity-dependent plasticity through high intensity, high repetition training programmes. There is a marked increase of elderly patients with SCI because of an increase in life expectancy. This form of rehabilitation is problematic and researchers are investigating novel treatment methods. Neural plasticity has been observed following intermittent hypoxia (IH) in respiratory and non-respiratory neurons. Only a single treatment showed a significant improvement in lower limb function. The study investigated if the conduction and excitability within ascending spinal tracts are influenced by IH. To assess this an electrical stimulus was delivered at the median or tibial nerve for 4 minutes. Three recordings were taken before, during and following the treatment. This creates somatosensory evoked potentials (SEPs) that were recorded using scalp electrodes. Nine healthy adults (21-35 yrs.) were exposed to the IH treatment which involves one-minute sessions of repeated exposures of hypoxic (FIo2= 0.09) and normoxic (FIo2= 0.21) air for 30 minutes. The oxygen saturation level (SpO2) did not drop below 96.6%. This indicated that for the subjects tested the IH treatment failed to produce the marked reduction in the O2 saturation that was observed in spinal cord injured patients. This explains why there was no significant difference in the peak to peak amplitude of the SEPs when comparing the values before with during and after IH (p > 0.05; two-tailed paired t-test). Furthermore, the heart rate and blood pressure were monitored and since the SpO2 level did not drop as low as 81% there was no significant change in blood pressure and heart rate following the treatment (p > 0.05; two-tailed paired t-test). Under the circumstances that the protocol was not effective we can conclude that it provides a way of creating a sham condition.

Understanding of respiratory syncytial virus (RSV) disease and vaccine development has been hampered by an incomplete understanding of the immune mechanisms of protection and disease in humans. Reinfection with antigenically similar strains of RSV occurs throughout life, and observational studies of natural infection do not permit analysis of events in the pre-symptomatic phase of disease. Experimental infection of healthy adults with a standardised inoculum was therefore used to study RSV upper respiratory tract (URT) disease. Pre-existing humoral immunity was assessed locally and systemically, and dynamics of URT symptoms, viral load and nasal mediators were analysed to identify factors involved in symptomatic infection. Forty subjects were enrolled of whom 24 developed infection and 17 exhibited typical cold-like features. Peak symptoms and viral load occurred on days 7 and 8 respectively. Pre-inoculation RSV-specific nasal IgA was most clearly associated with resistance to infection, but infection-boosted levels waned after 6 months. The frequency of acute IgG and IgA antibody secreting cells increased after infection and virus-specific IgG⁺ memory B cells (MBC) incremented around 4-fold. By contrast, IgA⁺ MBC were undetectable pre- and post- infection whereas influenza-specific IgG⁺ and IgA⁺ cells were found at comparable frequencies in convalescent blood from patients recovered from H1N1 influenza, suggesting a specific defect in IgA memory after RSV infection. Nasal viral load correlated with levels of nasal IFN-γ, CXCL10, MIG, TNF and IL-6. Subjects exhibiting early pre-symptomatic IFN-α responses showed reduced symptom scores and lower overall viral loads. Lifelong susceptibility to RSV infection thus reflects a specific defect in IgA memory to RSV, the cause of which needs to be determined. Symptomatic disease results from a failure of innate immune control of RSV replication in the pre-symptomatic stages of infection during the first 48 hrs of viral encounter. Viral replication leads to epithelial injury and inflammation reflected by a number of inflammatory mediators in nasal secretions. Mucosally-delivered vaccines could overcome the IgA memory defect and induce durable protection, which might be enhanced by inducing local IFN-α immediately after viral exposure.

Emphysema is characterised by airflow limitation that is a result of both loss of elastic recoil and small airways disease. It is poorly responsive to medical therapy. Lung volume reduction coils improve symptoms and lung function in the short term. However their mechanism of action and medium term effectiveness is not fully understood. Methods A randomised controlled study consisting of thirty patients with severe chronic obstructive pulmonary disease was performed. Control patients crossed over to the treatment arm at 12 months. The primary outcome was 6 minute walk distance at 12 months. Changes in spirometry, lung volumes, computed tomography measured lung volumes and gas trapping were also assessed. In a small subgroup of patients detailed physiological characterization was performed to assess changes in airways resistance, ventilation heterogeneity and lung elastic recoil. Results In the randomised study at 12 months, there was no significant difference in 6 minute walk distance between treatment and controls (between group difference 25m, 95% CI -40 to 59, p = 0.7028). There was a trend to improvement in symptoms measured by SGRQ score (-6.53 points, 96% CI -17 to 0.2, p = 0.0589) and significant improvements in FRC (-0.41L, 95% CI -0.86 to -0.1, p = 0.0077). Including the crossovers there were 4 patient deaths (13.3%). Target lobe volume at both inspiration and expiration was reduced with no overall change in gas trapping. Airways resistance by plethysmography did not change significantly. There was no significant change in elastic recoil. Conclusions Treatment with lung volume reduction coils is effective at reducing lung volume and may achieve its effect through volume loss. There could also be an effect through elastic recoil as there was a non-significant trend towards an increase after the intervention. There appears to be no effect on airways resistance. Careful patient selection is required as there is a risk of death following treatment.

No description available.

Fourteen smokers (S) underwent <SUP>99m</SUP>Tc-DTPA lung scans after refraining for 12 hours (chronic smoking, C) and after smoking until 1 hour before study (acute smoking, A). <SUP>99m</SUP>Tc-DTPA clearance was increased in C and further in A. Each smoker also underwent bronchoalveolar lavage (BAL) after either C or A and seven control non-smokers (NS) were also studied. Neutrophils in BAL were increased in A. Superoxide release (O<SUB>2</SUB><SUP>.-</SUP>) from mixed BAL leucocytes and plasma products of lipid peroxidation were increased in both smoking groups. In BAL fluid (BALF) and epithelial lining fluid (ELF) the latter doubled in C and increased 6 fold in A. Trolox equivalent anti-oxidant capacity (TEAC) decreased in plasma and increased in BALF. Reduced glutathione in the airspaces doubled in C. This increase was abolished in A. In a further study 15 healthy NS were exposed to filtered air (FA), or ozone 100 or 400ppb for 1 hour, during intermittent exercise. Ozone inhalation increased ELF volume overall. Neutrophils in BAL were increased 6 hours after ozone 400ppb compared to FA. Ozone inhalation decreased O<SUB>2</SUB><SUP>.- </SUP>release from mixed BAL leucocytes and products of lipid peroxidation in ELF, 1 and 6 hours after 400ppb. <I>In vitro</I> exposure of A549 cells to 1000ppb for 1 hour produced focal cell loss, with increased intracellular protein mixed disulphides, export of oxidized glutathione, and cytoskeletal disruption. In conclusion increased epithelial permeability and neutrophil influx in the airspaces, produced by oxidant inhalation are acute phenomena. In smokers they were associated with increased oxidant stress. In healthy non-smokers, in contrast, acute ozone inhalation was associated with decreased oxidant stress, at least as measured by the extent of lipid peroxidation and oxygen radical release from mixed BAL leucocytes, and in addition anti-oxidant levels were maintained. <I>In vitro</I> studies indicated the potential of ozone to increase oxidant stress in the respiratory epithelium and airspaces with profoundly damaging effects.