Background: Chronic obstructive pulmonary disease (COPD) constitutes a major cause of morbidity and mortality. Genome wide association studies (GWAS) have shown significant associations between airflow obstruction or COPD with a non-synonymous single nucleotide polymorphism (SNP) in the TNS1 gene (which encodes tensin1) with COPD. This SNP generates the amino acid change R1197W in the tensin1 protein. R1197W is associated with reduced FEV1. Aim: To examine tensin1 genotype, expression and function in human airways and cells from healthy controls and patients with smoking-related COPD or asthma. Methods: Lung resections were immunostained for tensin1. Tensin1 expression in cultured human airway smooth muscle cells (HASMCs) and bronchial epithelial cells (HBECs) was evaluated using qRT-PCR, western blotting and immunofluorescent staining. siRNAs were used to downregulate tensin1 expression, and cells from healthy subjects and patients were genotyped using restriction fragment length polymorphism (RFLP) analysis. Results: Immunohistochemical staining demonstrated increased tensin1 expression in the airway smooth muscle and lamina propria in COPD tissue, but not in asthma, when compared to healthy controls. Tensin1 was expressed in HASMCs and upregulated by TGFβ1. TGFβ1 and fibronectin increased the localisation of tensin1 to fibrillar adhesions. Tensin1 and α-smooth muscle actin (αSMA) were strongly co-localised in actin stress fibres. Tensin1 depletion in HASMCs attenuated αSMA expression and their contraction of collagen gels, but not proliferation. RFLP analysis revealed the presence of R1197W in predominantly people with COPD and asthma, but rarely in healthy controls. Conclusions: Tensin1 may promote airway obstruction by enhancing the expression of contractile proteins and their localisation to stress fibres in HASMCs. R1197W is associated predominantly with COPD and asthma suggesting that the polymorphism may promote dysfunction in HASMCs.

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COPD is a chronic, multifactorial lung disease with multi-organ, systemic presentation that affects approximately 20% of smokers. Diffuse parenchymal epithelial metaplasia is seen in COPD patients and although rarely described, is usually considered to be Lambertosis, a reparative extension of airway epithelium. Cellular phenotype and pulmonary distribution of diffuse metaplasia was examined histologically and the cells characterised as either rounded and uniform, large and irregular resembling macrophages or squamous and flattened. The lesions were found to be localised to the external adventitia of the airways and vessels, sub pleural zone, hilar regions, ectopic fibrotic deposits and the external facet of alveolar epithelium. This distribution is not consistent with Lambertosis. This repair process hypothesis was evaluated by cellular markers consistent to known airway progenitors. No expression of CK5/6 basal nor CC10 Clara cell was detected. Surfactant presence suggested a type II phenotype indicating an alveolar origin. These findings further refute Lambertosis. The metaplastic population was further characterised using epithelial and progenitor markers. These cells displayed occasional multipotent, progenitor markers possibly originating from bone marrow or resident stem cells (CD34/CD133). The cytokeratin profile consistent with simple epithelium suggested new cell colonisation and mixed cytokeratin phenotype indicated a failure to mature. In addition to this evidence of repair response, metaplastic cells were found on expanded mesenchyme. This remodelled, scarred tissue further indicates damage and repair. Mutation detection of EGFR L858R by PCR did not show molecular changes indicative of early change in some adenocarcinoma, suggesting these cells do not progress to pre-neoplastic. From this, we can conclude that diffuse metaplasia in COPD peripheral lung is not consistent with Lambertosis. Overall, characteristics including surfactant and mucin presence, suggest a primitive epithelial phenotype, potentially a result of damage and aberrant repair.

<i>In vitro </i>models were developed to measure cytokine responses to various agents implicated in COPD. These examined the interaction, antagonistic, indifferent, additive or synergistic, between cigarette smoke and infectious agents or their products on cytokine production. <i>Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis </i>and respiratory syncytial virus are common bacterial and viral pathogens isolated from this group of patients. A human monocyte cell line in a model provided a consistent means to examine these interactions, and human peripheral blood monocytes from blood donors were used to study the individual variations in the responses. Effects of virus infection on bactericidal activity of human monocytes common bacterial respiratory pathogens were also examined. An epithelial cell line and monocytes were investigated for the effects due to virus infection on expression of some of the surface antigens relevant to bacterial binding and immune response. The agents used in the study elicited inflammatory responses that could contribute to damage to the respiratory tract and these individual factors could be more harmful in combination. Monocytes from only a proportion of individuals exhibited extreme responses to these agents signifying the role of individual genetic make up in inflammatory processes. Virus-infected monocytes significantly decreased their ability to bind and kill bacteria. Compared with uninfected cells, fewer bacteria bound to virus-infected cells and intracellular bactericidal activity was also decreased. Exposure to a number of harmful factors for longer periods in individuals with certain genetic profiles for inflammation may cause significant damage to the respiratory tract resulting in COPD, and exacerbation in its course.

Once thought to be an inflammatory-fibrotic disease, idiopathic pulmonary fibrosis (IPF) is regarded as an epithelial-fibrotic phenomenon with inflammation increasingly viewed as an epiphenomenon. Markers indicative of disruption in epithelial integrity have been associated with a worse outcome in IPF. However, these associations have only been minimally explored in the interstitial lung disease of systemic sclerosis (SScILD). This project tested the hypothesis that progression of SSc-ILD is linked to breaches of lung epithelial integrity. The aims were: 1) To study the prognostic significance of epithelial markers including 99mTc_DTPA pulmonary clearance and serum KL-6 levels and compare with other known prognostic markers. 2) To examine relationships between inhaled 99mTc_DTPA clearance and: a) histological features, b) bronchoalveolar lavage, in particular eosinophils, c) serum KL-6 levels. When traditional prognostic measures (HRCT, PFT, BAL) were assessed in SSc-ILD, baseline DLco levels, the extent of disease on HRCT and the extent of a reticular pattern on HRCT were all strong predictors of mortality. The extent of a reticular pattern on HRCT was the strongest predictor of disease progression. However, 99mTc_DTPA clearance was the strongest predictor of more progressive disease, surpassing the value of HRCT and PFT. Serum KL6 levels were predictive of more progressive disease but not of survival. BAL eosinophilia and/or neutrophilia have traditionally been viewed as markers of progression. Indeed, a BAL neutrophilia was found to be predictive of increased mortality in SSc-ILD but interestingly, this association was not significant after disease severity was controlled for. However, there was a significant correlation between 99mTc_ DTPA clearance and serum KL6 levels, and between 99mTc_DTPA clearance and BAL eosinophil levels. The profibrotic effects of eosinophils and their interactions with normal lung fibroblasts were further investigated by determining the contribution of profibrotic cytokines such as TGFp. Functional studies looking at the effects on pathologic indicators of fibrosis such as extraceullar matrix production and fibroblast proliferation were also performed Collectively, these results suggest that disruption to the epithelium may playa pivotal role in pathogenesis in SSc-ILD. Thus the use of 99mTc_DTPA pulmonary clearance may have a role in routine clinical practice. Perhaps of more practical use is the measurement of serum KL-6 levels, which is easily performed with no risk to the patient.

Respiratory tract infections are a leading cause of morbidity and mortality worldwide. Although the classical manifestations of respiratory tract infections are defined, the pathological cause responsible for disease is not often identified. Human metapneumovirus (hMPV) causes a spectrum of illnesses ranging from asymptomatic infection to severe bronchiolitis. Little is yet known about the pathophysiology of hMPV infection, however, it is similar to the related pneumovirus and human respiratory syncytial virus (hRSV). Since hMPV appears to cause disease similar to that of hRSV, one can hypothesize that hMPV also causes similar extra-pulmonary manifestations. The objectives of this thesis were divided into three main areas; prevalence / diagnosis, immune response, and role of hMPV in extra-pulmonary manifestations. This study reports, for the first time, the prevalence and clinical picture of hMPV infection in the Maltese community. Moreover, it suggests that the use of detection assays such as real-time PCR would be very helpful in the diagnosis and early treatment of viral respiratory pathogens, such as hMPV especially in pediatric and immunocompromised patients. This study also suggests that multiplex RT-PCR assays reduce the turnaround time and have higher detection rates than shell vial cultures. Up to now, there have been no confirmed reports of hMPV causing disease outside the respiratory tract. Although a positive hMPV result in a subject with documented myocarditis suggests that hMPV was the sole etiological agent, the association of this virus with myocarditis was not statistically significant. Nevertheless, to investigate this potential relationship further large prospective studies are needed.

Rationale: Epithelial ciliary dysfunction is a feature of asthma that is believed to contribute to persistent symptoms and recurrent exacerbations. However, the mechanism underlying this dysfunction is unknown. Hypotheses: The ciliary dysfunction of asthmatic airway epithelial cells is due to an intrinsic abnormality that leads to a high susceptibility of these cells to an environmental challenge, which results in a chronic inflammation and thus a predisposition of asthma exacerbations. Methods: Primary airway epithelial cells from basal cells and ciliated cultures, and fresh ciliated strips, were used. Baseline protein and gene expression were assessed by protein quantification and microarrays. Ciliary function was studied using video-microscopy. Asthmatic sputa were used as the environmental stimuli; microbiology was assessed. Oxidative stress and the role of NADPH oxidase (NOX) 4 were assessed using immunohistology, reactive oxygen species (ROS) quantification, quantitative gene expression, and the NOX1/4 inhibitor GKT137831. Results: In ex vivo ciliated cultures, ciliary dysfunction did not persist but was evident in cells from asthmatics following asthmatic sputum inoculation. Bacterial 16S load increased equally in asthmatic and control samples. Oxidative burden in asthmatic bronchial epithelium was increased and was related to the percentage of sputum neutrophils. NOX4 expression and hydrogen peroxide-induced intracellular ROS generation were significantly elevated in epithelial cells from neutrophilic subjects, with the latter being attenuated by NOX4 inhibition. In asthmatic ciliated cells obtained directly from bronchoscopy, inhibiting NOX4 markedly improved ciliary function and was related to the intensity of neutrophilic inflammation. Summary: The up-regulation of NOX4 expression that is evident in asthmatic ALI cultures might promote the susceptibility of the bronchial epithelium to the development of ciliary dysfunction in the presence of an abnormal microenvironment, implicating NOX4 as a potential therapeutic target for neutrophilic asthma. An increase in the sample size is required to increase the strength of this conclusion.