C3a and C5a anaphylatoxins are proinflammatory polypeptides released during complement activation. They exert their biological functions by interacting with the G protein-coupled receptors, C3aR and C5aR respectively. Activation of the complement system has been implicated in the pathogenesis of many inflammatory diseases including asthma. Little is known, however, about the expression and location of complement components in asthmatic airways. -- Experiments described in this thesis demonstrate the expression and localisation of certain complement components and their two receptors in the bronchial mucosa. C3a and C5a-stimulated production of remodelling mediators and the biological function of the anaphylatoxins on the structural cells was also assessed. -- Immunohistochemical analysis revealed elevated expression/deposition of C3 and C5 components in the epithelium, airway smooth muscle and submucosa of asthmatics compared with controls, and also demonstrated expression of the two complement receptors on airways structural cells, including airway epithelial cells, endothelial cells, fibroblasts and smooth muscle cells (SMC). -- In general C3a was the more effective of the two anaphylatoxins in inducing structural cellular proliferation: C3a increased fibroblast and endothelial cell proliferation at a range of concentrations embracing the physiological, although it increased SMC proliferation only at the highest concentration (10~7 M) employed, and epithelial cell proliferation only at the lowest concentration (10~9 M) employed. C5a induced fibroblast proliferation but had no effect in this regard on the other structural cells.

Background: Chronic obstructive pulmonary disease (COPD) represents a spectrum of disorders encompassing chronic bronchitis and emphysema, associated with cough, excess mucus and exercise-related dyspnoea and characterized by a progressive reduction in airflow. Development of these symptoms is associated with chronic exposure to noxious particles or gas, most commonly from tobacco smoking, which triggers abnormal inflammation in the lung. Consistent with this increased inflammatory burden, oxidative stress has been demonstrated in COPD patients, largely through determination of antioxidant and oxidative damage marker concentrations in exhaled breath condensate and induced sputum. Whilst these samples are easier to obtain than bronchoscopy-based lavage, there remains contention concerning how well they reflect the distal airway lining fluids and hence the actual disease state. In the present study, I therefore investigated the oxidative status of bronchoalveolar lavage fluids (BAL) from well defined groups of COPD patients; current and ex-smoker, as well as aged and smoking matched controls. In addition, as COPD has been argued to be a disease of accelerated ageing a group of young controls was included to examine the extent to which age influences the endpoints under consideration. Chapter 2: In the first experimental chapter I investigated the evidence for oxidative stress in the airways of subjects with COPD (smokers and ex-smoking) relative to age and smoking matched controls. In addition, young healthy non-smokers and mild asthmatics were included to investigate the impact of age on the examined parameters, as well as to compare antioxidant defences in the context of acute and chronic inflammation. In this chapter, I examined respiratory tract lining fluid antioxidants sampled in bronchoalveolar lavage fluid, focusing on low molecular weight antioxidants (glutathione, urate and ascorbate) and their oxidation products (glutathione disulphide and dehydroaspresence) of COPD, but observed smoking-related increases in glutathione and ferritin, and age-related increases in dehydroascorbate and 4-hydroxy-2-nonenal. Chapter 3: In the second experimental chapter I attempted to understand the age-related increase in oxidation markers observed in the lavage samples from chapter 2. Pro-oxidant metal (Fe and Cu) concentrations were determined in the lavage samples from each of the groups used in chapter 2, by inductively coupled plasma mass spectrometry and a novel assay was developed based on metal catalysed oxidation of ascorbate, to provide a functional measure of the catalytically active metal pools at the surface of the lung. Through the use of selective chelators, the relative contribution of labile Fe and Cu pools was assessed. In this chapter I was able to demonstrate that respiratory tract lining fluid Cu concentrations increased with age, in parallel to an increased pro-oxidant status in the RTLF and evidence of a non-transferrin bound Fe pool. These indices appeared related to the concentration of oxidation markers reported in chapter 2. Chapter 4: Given the failure to detect increases in gross measures of oxidative damage in COPD patients, it was decided that the focus should shift toward a more refined focus on specific oxidations to proteins functionally related to the pathogenesis of the disease. I decided based on the pre-existing literature, that there was merit in focusing on 4-hydroxy-2-nonenal adduction of proteins. To achieve this, I attempted to develop a mass spectrometry-based method for the identification of adducted proteins and protein sequences. This was an ambitious undertaking and unfortunately I was only able to take these experiments so far, but at least was able to demonstrate the feasibility of the method. The methodology developed was sufficiently promising to warrant further investigation after the completion of my PhD. Short conclusion: In this study I was unable to demonstrate any evidence of oxidative stress in the airways of patients with COPD, either ex- or current smokers. I did however, observe evidence of increased oxidative damage, catalytic metal (Cu) concentrations and pro-oxidant metal activities (Cu and non-transferrin bound Fe) with age. At the end of this thesis, despite the attempt to investigate markers of oxidative stress in a relevant compartment (the distal lung) and in carefully controlled groups (aged and smoking matched), the role of oxidative processes in COPD remains oblique.

Asthma is a chronic inflammatory disease of the airways. The current cornerstone for asthma treatment is glucocorticoids (steroids), and the majority of patients respond to steroids with an improvement in lung function (Steroid Sensitive; SS). However a proportion of asthmatics do not respond to steroids with improvement in lung function (Steroid Resistant; SR), who represent those patients most at risk. Epidemiological studies demonstrate that asthma severity and poor responsiveness to treatment, is associated with vitamin D insufficiency and deficiency. Vitamin D is an immunomodulatory molecule which induces the synthesis of antimicrobial peptides and anti-inflammatory molecules, and inhibits pro-inflammatory cytokine synthesis. The overall goal of this work was to investigate immunological differences between SS and SR asthma patients, and effects of vitamin D that may explain the epidemiological observations. Peripheral blood from a clinically well-characterized cohort of SS (n=14; mean improvement in lung function or FEV1 post 2-weeks oral prednisolone 16.1%) and SR (n=23; no improvement in FEV1) asthmatics was studied by ex vivo flow cytometry. Major findings included evidence for significantly higher frequency of myeloid dendritic cells, and lower frequency of Foxp3+ CD4+ T cells in the peripheral blood of SR as compared to SS asthmatics. The accepted measure of vitamin D status is serum 25-hydroxyvitamin D3, 25(OH)D, and the severe asthmatics had significantly lower levels of serum 25(OH)D as compared to healthy controls. A significant positive correlation between the number of Foxp3+ T cells in the peripheral blood and serum 25(OH)D was observed. Following culture of PBMCs with anti-CD3 activation it was found that severe asthmatics synthesised significantly higher IL-17A levels than healthy controls, which was most striking in the SR patients. The steroid Dexamethasone (Dex) enhanced IL-3 17A production in culture, whereas the active form of vitamin D 1,25-hydroxyvitamin D3 (1,25(OH)2D3) significantly reduced IL-17A production in a Dex-independent manner. This work proposed that IL-17A inhibition by 1,25(OH)2D3 may be partially due to a CD39-dependent mechanism. In vivo evidence for a positive association between serum 25(OH)D and Foxp3+Treg numbers was further investigated in vitro. High concentrations of 1,25(OH)2D3 (10-6M) increased the frequency of Foxp3+ T cells in culture through two main mechanisms: the induction of IL-2, on which Foxp3+Treg are dependent, as well as less inhibition of Foxp3+ over Foxp3- T cell proliferation. Lower, and likely more physiological concentrations of 1,25(OH)2D3 (10-7M), which are known to enhance IL-10 synthesis, failed to significantly increase the frequency of Foxp3+ CD4+ T cells. However upon modulation of the cytokine environment to one of low IL-10 and high TGFβ, this concentration of 1,25(OH)2D3 significantly increased Foxp3+ Treg frequency. Collectively this data highlights additional immunoregulatory properties of 1,25(OH)2D3 including induction of anti-inflammatory cytokines such as IL-10 and TGFβ, inhibition of pro-inflammatory cytokines such as IL-17A and IL-22 as well as the direct induction of Foxp3+ Tregs. In conclusion evidence for a number of immunomodulatory effects of 1,25(OH)2D3 exists that may help to reduce uncontrolled inflammatory responses associated with severe asthma.

S. pneumoniae (pneumococcus) is a major cause of pneumonia, sepsis, and meningitis, responsible for over 1.2 million deaths per year. Rates of antibiotic resistance are rising, with over one third of isolates in the US and parts of Europe showing a reduced susceptibility to penicillin. Although pneumococcal conjugate vaccines have resulted in a decline in invasive disease caused by the pneumococcal serotypes included in the vaccine, non-vaccine serotypes have been shown to cause replacement disease. To address these important clinical challenges and provide cross serotype protection against pneumococcal infection, alternative therapies are urgently needed such as the exploitation of bacteriophages, which can specifically target and kill pneumococci. Bacteriophages are being developed to treat a range of bacterial pathogens due to their ability to kill pathogens which are resistant to conventional antibiotics, and due to their specificity and ability to access and replicate in difficult micro-environments within the human body. Although the previously isolated pneumococcal lytic phages; Dp-1 and Cp-1 showed promise as a treatment for pneumococcal infection, they could only infect non-capsulated strains, which are attenuated and non-invasive in the human clinical setting. This project describes the isolation and characterisation of a new lytic phage SP-QS1, which can infect and significantly reduce the load of capsulated pneumococcal strains that cause human invasive disease. SP-QS1 is a distinctive new siphovirus with prolated-head, non-contractile tail and tail fibres. The interaction between SP-QS1 and S. pneumoniae in both in vitro and in in vivo assays demonstrated that it is able to significantly reduce the amount of S. pneumoniae in two mouse models of invasive pneumonia; the intranasal and the intravenous model of infections. The genomic sequencing of SP-QS1 revealed that genes with recognisable homologies are often ordered according to the following; genes involved in phage packaging, structural proteins, replication and genes associated with cell lysis. Interestingly, SP-QS1 genome does not encode CRISPR sequences, proteins with trans-membrane domains or regulatory elements. In addition, because the phage genome does not encode integrase genes, it appears to be a genuine lytic phage. Genetic characterisation of SP-QS1 genome illustrated that this phage encodes for glycosyltransferase, and it is suggested to be responsible for capsule degradation. SP-QS1 shows promise to control and treat the infections caused by S. pneumoniae.

No description available.

Mucin hypersecretion in the airways is a hallmark of Chronic Obstructive Pulmonary Diseases (COPD) and in particular of chronic bronchitis. This study investigates how bacterial products such as LPS affect mucin production (the main constituent of mucus) and how surfactant phospholipids may modulate this effect. In two human bronchoepithelial cell lines, NCI-H292 and Calu-3, both of which express mucin gene and secrete mucins, LPS treatment resulted in an increase in secretion, production and of gene expression of MUC5AC, one of the main mucins present in airways secretion, and of MUCI, a membrane anchored mucin. LPS treatment also increased the presence of fucose, sialic acid and N-acetylgalactosamine, the main elements involved in mucin glycosylation. Using fluorescent microscopy, TLR4, part of the complex receptor for LPS, was detected on the surface of these cells and CD74, a co receptor for LPS, detected internally. Following LPS exposure, CD14 was released from cells and was detected colocalised with TLR4. LPS induced NFкB activity and the utilisation of a NFкB inhibitor abolished the effect of LPS on mucin gene expression. The main surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPC), inhibited the effect of LPS on mucin production and glycosylations. It reduced NFкB activity induced by LPS but had no effect on CD14 release. It also inhibited mucin gene expression induced by IL-lβ and by TNF-α. Other surfactant phospholipids were tested: palmitoyl-linoleoyl-phosphatidylcholine (PLPC) had a similar modulatory effect to that seen with DPPC, palmitoyl-arachidonoylphosphatidylcholine (PAPC) showed no effect whereas palmitoyl-oxovaleroylphosphorylcholine (POVPC), a product of oxidised PAPC, also inhibited the activity of LPS. These results suggest that bacterial products in the airways are capable of inducing mucin secretion and that surfactant phospholipids modulate this effect. These findings indicate a therapeutic role for surfactant supplementation in chronic respiratory diseases.

The manuscripts submitted in this thesis describe the early development and implementation work for a molecular technology based service for the diagnosis of respiratory viral infection in Wales. The service allowed for a greater understanding of the spectrum of disease caused by respiratory viruses as molecular methods were proven rapid and sensitive when compared to traditional laboratory methods. This work transformed the understanding of respiratory virus infection in both vulnerable and healthy patients in the community and hospital setting and eventually informed on the most appropriate way to deliver a 2lst century respiratory virus diagnostic service. Implementation of the service has had a direct impact on public health by improving the detection and monitoring of respiratory virus outbreaks and by providing information regarding the circulation of respiratory viruses in the community. The service in Cardiff became part of a network of laboratories that responds to new and emerging respiratory viral infections across the UK and the expertise gained proved instrumental in the Welsh laboratory response to the influenza A (H1N1) 2009 virus pandemic and the issues that arose as a direct consequence including an oseltamivir resistant outbreak. Ongoing developments in the field of molecular diagnostics together with the increasing repertoire of respiratory viruses means that the service is constantly improved to ensure that Wales continues to have a first-class service for the diagnosis of respiratory viral infection.

Asthma is a chronic inflammatory disease characterised by reversible airflow obstruction. Based on the relationship between a lack of exercise and chronic diseases, the latest guidelines from the Department of Health (DH) recommend physical activity across the whole population (DH, 2011). Exercise Induced Bronchoconstriction (EIB) is a 'sub-type' of asthma which affects approximately 90% of all individuals with asthma and an additional 10% of the healthy normal population (ATS/ACCP, 2003; Anderson & Kippelen, 2012); thus, EIB may be an important limiting factor for physical activity and an important 'barrier to exercise' for a number of individuals. Asthma is identified primarily by the occurrence of symptoms (wheezing breathlessness and dyspnoea), peak expiratory flow rates (PEF) and spirometry (Pulmonary Function Tests - PFT). The current spirometry guidelines for the characterisation of asthma include a fixed criteria for the ratio between forced expiratory volume in one second and forced vital capacity (FEV1/FVC) (Miller et al., 2005b). This fixed criteria approach lacks specificity and is likely to misdiagnose approximately 20% of patients (Miller et al., 2011). The American Thoracic Society (ATS) and the European Respiratory Society (ERS) guidelines have acknowledged these concerns and have issued position statements for the use of a different approach using a 'lower limit of normality' (LLN) derived from a matched healthy population (Miller et al., 2009). Based on the fixed criteria, it has been shown that there is under diagnosis of participants with mild-moderate symptoms participants in the younger age group (Cerveri et al., 2009; Hansen et al., 2007; Miller et al., 2011; Roberts et al., 2006; Swanney et al., 2008). The currently available pharmacological therapies for asthma and EIB are effective (corticosteroids and bronchodilators), however long-term usage of these medications is associated with issues of tachyphylaxis and negative side effects (Barnes, 2010; GINA, 2011). There is some evidence from observational and intervention studies to suggest a beneficial effect of fish oil (comprising of omega-3 (n-3) polyunsaturated fatty acids (PUFAs)) in inflammatory diseases, (specifically asthma). Marine based n-3 PUFA have therefore been proposed as a possible complimentary/alternative therapy for asthma. The proposed anti-inflammatory effects of fish oil may be linked to a change in cell membrane composition. This altered membrane composition following fish oil supplementation.

This research investigated the use of acoustic and electroglottographic (EGG) analysis in support of the diagnosis of pathologies affecting the larynx and voice production. Experiments were conducted (1) to verify the reliability of perturbation measures from sustained vowels and (2) to obtain normative perturbation values in a dysphonic population. The studied measures included jitter, shimmer, signal-to-noise ratio, EGG closed-quotient, and EGG speed index. Although such parameters have been widely studied in the field of voice production, their reliability has been investigated mostly on non-dysphonic speakers, synthetic signals, or artificially produced perturbations. Synthetic stimuli may permit well-controlled systematic studies, but still fail in adequately reproducing the vocal perturbations found in dysphonic speakers. Therefore, many reported normative values and computational algorithms need to be validated on real disordered voices before being recommended for clinical use. Some computational algorithms are being proposed in this thesis, including a robust and reliable method for measurement of acoustic jitter, a procedure for estimating the signal-to-noise ratio on a cycle-to-cycle basis, and a highly accurate EGG fundamental frequency tracker. Practical protocols for data acquisition in a clinical environment are also being proposed. The most important contribution of this research was the development of a method for detecting certain asymmetries in the posterior part of the larynx. These problems were commonly found in the "no-abnormality-detected" and "functional" diagnostic groups. The detection method exploits a differential increase in EGG jitter values that was induced, possibly, by instabilities in the positioning of the arytenoid cartilages during phonation. These asymmetries, which can be caused by unilateral paralysis of the superior laryngeal nerve, among other causes, seemed particularly harmful to singers and other professional voice users.

Introduction: A number of epidemiological studies have shown a significant association between asthma and work as cleaner but reporting schemes and workforce surveys have identified typical features of occupational asthma in only a small minority of cleaners. This discrepancy is due either due to under-reporting by clinician; misattribution of work-exacerbated asthma or other respiratory disease by the epidemiological studies, or the development of occupational asthma with atypical symptoms that make it identifiable epidemiologically but difficult to diagnose clinically. Hypothesis: The study hypothesis is that cleaners’ asthma is induced by chronic low-level irritant exposures that gradually induce airway hyper-responsiveness but do not cause work-related airway constriction/symptoms. It is thus identified by epidemiologists but is not easily identifiable clinically. Aim: The aim of this PhD is to identify the proportion of cleaners with feature of occupational asthma and to identify risk factors for cleaners’ asthma.