Rationale Cardiovascular disease (CVD) is a leading cause of mortality in patients with COPD. Aortic stiffness, measured using aortic pulse wave velocity (PWV), an independent, non-invasive, predictor of CV risk; and inflammatory markers are increased in COPD. Screening tools for community based identification of increased CVD risk, and a proactive approach to addressing primary prevention of CVD is needed. Statins modulate aortic stiffness and are anti-inflammatory, but are not currently used for primary prevention in COPD. Objectives Proof of principle double-blind Randomised Control Trial (RCT) to determine if six weeks simvastatin 20mg od reduces aortic stiffness, systemic and airway inflammation in COPD. Cross-sectional pilot study comparing a non-invasive measure of oxidative stress (skin “AGE”) in COPD and controls, to lung function and aortic stiffness. Methods Stable patients (n=70) were randomised to simvastatin or placebo treatment. Pre- and post-treatment aortic stiffness, blood pressure, spirometry, circulating inflammatory mediators and lipids were measured; airway inflammatory markers were performed where possible. Predefined subgroup analysis was performed where baseline aortic PWV >10m/s. For the cross-sectional study stable COPD patients (n=84) and controls (n=36) had lung function, arterial stiffness and skin AGE measured. Results In the RCT the active group achieved significantly lower total cholesterol, but no significant drop in aortic PWV compared to placebo group: -0.7(95%CI -1.8,0.5)m/s, p=0.24; or inflammatory markers. In those with higher baseline aortic PWV, n=22, aortic PWV improved in the active group compared to placebo: -2.8(-5.2,-0.3)m/s, p=0.03. Skin AGE was increased in COPD compared to controls, inversely related to lung function, and directly related to aortic stiffness. Conclusions We could not detect any significant difference in the change in aortic PWV in patients with COPD taking simvastatin compared to placebo. We did, however, report a significant and clinically relevant reduction in aortic PWV in those with high baseline aortic stiffness, suggesting a potential for statins to reduce CV morbidity in high risk individuals. The pilot cross-sectional study suggests there is an indication to assess the potential role of skin AGE in patients with COPD as a non-invasive measure of CV risk.

Macrolide antibiotics, including Azithromycin (AZM), can improve clinical symptoms in asthma regardless of infection status. Mechanisms underlying these beneficial effects are yet to be fully elucidated. Asthma is associated with a defective airway epithelium with reduced expression of structural proteins and aberrant repair responses. In vitro, AZM has shown anti-inflammatory and anti-viral actions, as well as enhancement of airway cell barrier integrity. Therefore, it was hypothesised that the beneficial effects of AZM in asthma may involve barrier reinforcement. The main aims were to determine the effects of AZM on airway epithelial function in vitro, in vivo and ex vivo. Primary normal human bronchial epithelial cells (HBEC) were differentiated in vitro through an air liquid interface. Severe asthma patients were administered 250mg daily AZM for 6 weeks, with clinical outcome measures and bronchoscopy pre- and post-AZM. Addition of AZM to HBEC in vitro enhanced the development of a differentiating epithelial barrier over 14 days, which was accompanied by reduced permeability, increased thickness, reduced mucin expression and suppressed endogenous release of MMP-9. Importantly, MMP-9 levels inversely correlated with barrier integrity, providing a putative mechanism. Clinical measures from 10 asthma patients were heterogeneous both pre- and post-AZM. Overall, symptoms, lung function and inflammation did not significantly alter and there was no association between clinical measures and the epithelial barrier of bronchial biopsies. The current findings suggest that AZM aids in HBEC barrier formation in vitro. This novel finding may relate to the beneficial effects of AZM reported in vivo e.g. through reducing susceptibility to damage and inflammation during re-epithelisation. This could not be confirmed in vivo due to the low number of samples obtained. The current findings add further evidence towards the beneficial non-antibacterial effects of AZM and may have implications for the prospective targeting of the epithelium for clinical benefit in asthma.

The work described within this thesis was conducted at the University of Nottingham between April 2011 and March 2014. Due to the inter-disciplinary nature of this work it was undertaken by the author in conjunction with the other scientists in the Translational Imaging group at the Sir Peter Mansfield Magnetic Resonance Centre, University of Nottingham and collaborators in both the Pulmonary Biology group, University of Nottingham and the Respiratory Pharmacology group, Imperial College London. Pulmonary hyperpolarized (hp) noble gas magnetic resonance imaging (MRI) has seen increasing development and utility over the past two decades. However the application of this relatively new pulmonary imaging modality to small animal models is technically challenging. Ex vivo lung models have allowed for the investigation of functional respiratory measurements in small animals but have yet to be utilized with hp noble gas MRI. The ex vivo lung model presented within this work allowed for the study of pulmonary physiology using hp 129Xe and hp 83Kr MR imaging in intact lungs from both healthy rodents and rat models of respiratory disease. Novel hp 129Xe imaging protocols were developed to provide measurements of functional respiratory parameters and to gather information of regional gas distribution in healthy excised rodent lungs. Furthermore the developed 129Xe methodology was used to study regional responses in an ex vivo model of human asthma after intravenous deliveries of increasing quantities of the bronchoconstricting agent methacholine. The ex vivo model provided the platform to develop the novel lung imaging technique of hp 83Kr surface quadrupolar relaxation (SQUARE) MRI with this new methodology used to study an excised rat model of emphysema potentially providing the first application for this quadrupolar noble gas isotope in the field of respiratory medicine.

Master of Science / Department of Kinesiology / Craig A. Harms / An increased work of breathing during heavy whole body exercise can lead to respiratory muscle fatigue (RMF) and decreased leg blood flow. Heavy exercise also increases inactive limb and cutaneous blood flow. It is not known, however, how RMF affects inactive limb and cutaneous blood flow. Therefore, we tested the hypothesis that RMF during heavy exercise would reduce: 1) inactive limb blood flow, 2) inactive limb vascular conductance, and 3) inactive limb cutaneous blood flow. Twelve healthy men (23 ± 2 yrs) completed baseline pulmonary function tests followed by an incremental cycle test to VO[subscript]2[subscript]max. Subjects then cycled at both 70% and 85%VO2max (randomized) for 20 minutes. Subjects performed a second 85%VO[subscript]2[subscript]max test ingesting N-acetylcysteine (NAC) (1800mg), which has been reported to reduce RMF, 45 minutes prior the test. Maximum inspiratory pressures (P[subscript]Imax) were measured prior to and immediately following each exercise trial to determine RMF. During exercise, brachial artery blood flow (BABF) was measured via Doppler ultrasound and arm cutaneous blood flow was assessed by laser-Doppler flowmetry. Cutaneous vascular conductance (CVC) was calculated as flux/mean arterial pressure and scaled as % maximal CVC (sites heated to 46[degrees]C). Mean arterial pressure (MAP) was measured manually. Significant RMF occurred with 85%VO[subscript]2[subscript]max (12.8 ± 9.8%), but not with 70%VO[subscript]2[subscript]max (p>0.05). BABF significantly increased from baseline to end exercise in both conditions and was significantly lower (~18%) following the 85%VO[subscript]2[subscript]max test. The amount of RMF at 85%VO[subscript]2[subscript]max was inversely related to the change in BABF (r= -0.66, p<0.05). BA vascular conductance was significantly higher at end exercise at 70%VO[subscript]2[subscript]max compared to 85%VO[subscript]2[subscript]max (2.60 ± 0.73 vs. 2.00 ± 0.42 mLmin[superscript]-1mmHg[superscript]-1, resp.). The amount of RMF at 85%VO[subscript]2[subscript]max was inversely related to BA vascular conductance at end exercise (r= -0.80, p<0.05). Cutaneous vascular conductance was not different (p>0.05) between trials. With NAC, RMF was reduced and BABF was consequently significantly higher (~30%) compared to 85%VO[subscript]2[subscript]max. These data suggest that RMF during heavy whole body exercise decreases inactive arm blood flow and vascular conductance, but not cutaneous blood flow.

The purpose of the study was to determine the adherence to and usage of the N95 respirator mask as a TB control measure in healthcare facilities in the Tshwane district during managing a patient suspected or confirmed with pulmonary TB infection. A cross-sectional, descriptive survey design was used to collect the data. A sample of 204 healthcare professionals and healthcare workers working in five healthcare facilities in the Tshwane health district met the inclusion criteria for this study. More than 83.33 %( n=170) of the staff were nurses; physiotherapists in the healthcare facility constituted the lowest percentage (1.96%; n=4). Chi-square test was done to test whether there was a significant difference between knowledge on the fit test of the N95 respirator mask and the healthcare professionals and healthcare workers. There was no significant difference in knowledge across the five staff categories P-value of >0.05. Ensuring successful protection of the healthcare workers from contracting M-TB in the TB healthcare facilities depends on effective implementation of a respiratory protection programme and the correct usage of the N95 respirator mask.

Introduction: Vitamin D is a micronutrient that possesses immunomodulatory actions. Higher vitamin D status has been associated with decreased incidence of acute respiratory infections (ARI) in a number of observational studies. However, mechanistic in vitro work investigating effects of vitamin D on the immune response to ARIs is lacking, especially for rhinovirus, which is the most common respiratory pathogen. Results of clinical trials of vitamin D supplementation in the prevention of ARIs have also been conflicting, in that some demonstrate a protective effect of this intervention against ARI, while others do not. Methods: An immunological assay of ex vivo stimulation with TLR ligands and pathogens in blood samples from participants with asthma, COPD or neither condition in three randomised controlled trials of vitamin D supplementation for the prevention of ARI and exacerbations was developed. This assay was used in conjunction with cellular profiling of clinical trial blood and sputum samples, and a rhinovirus-infected human alveolar cell line (A549 cells) to determine the effects of vitamin D in the protection against acute respiratory infections. Results: The main finding of cell culture experiments was that A549 cells pre-treated with physiological concentrations of 25-hydroxyvitamin D (25[OH]D, the major circulating vitamin D metabolite) had increased resistance to rhinovirus infection, which was associated with attenuation of rhinovirus-induced intercellular adhesion molecule-1 (ICAM-1) and platelet-activating factor receptor (PafR) expression. Immunological analysis of clinical trial samples did not demonstrate any consistent effect of bolus-dose vitamin D supplementation on circulating or pathogen-stimulated inflammatory profiles, or on inflammatory indices in induced sputum. Conclusions: Co-incubation with 25(OH)D was associated with transient protection against rhinovirus infection in a respiratory epithelial cell line in vitro, but these findings did not translate to any changes in cellular profile or inflammatory mediator release in clinical trials samples following in vivo vitamin D supplementation.

Background: Idiopathic Pulmonary Fibrosis (IPF) is a physiologically devastating disease. The debilitating nature and high mortality rates make this one of the most lethal conditions, usually associated with median time to mortality of around 3 years. Increased deposition of extracellular matrix (ECM) and fibroblast accumulation are hallmarks of idiopathic pulmonary fibrosis (IPF). We hypothesise that the ECM in IPF is structurally abnormal by virtue of aberrant cross linking and promotes fibroblast accumulation. This study examined the structure and biological activity of IPF derived ECM and how this related to the expression of ECM cross linking enzymes as well as how inhibiting Transglutaminase 2 affects active fibrosis in the murine Bleomycin model. Methods: Primary fibroblasts from 3 patients with IPF and 3 controls were isolated from biopsy samples and characterised by immunocytochemistry. ECM from these cells was deposited onto tissue culture plastic, cells removed using ammonium hydroxide and confirmed by electron microscopy (SEM). IPF and control cells were then grown on their own ECM or ECM derived from other cells. ECM was labelled with 3H-proline and digested with recombinant proteases and tritium liberation counted by scintillation as a measure of collagen proteolysis. A pilot study was carried out where C57BL/5J mice received a single intratracheal instillation of Bleomycin (2mg/kg) and administered cystamine dihydrochloride by intraperitoneal injection (IP), once a day for ten consecutive days at 40mg/kg or 100mg/kg, at 3 different time points. Results: IPF derived fibroblasts had more distinct organisation of fibrous matrix filaments on the cell surface and between adjacent cells by SEM. Both control and IPF lung fibroblasts expressed transcripts for lysyl oxidase (LOX), LOXL1, LOXL2, LOXL3, LOXL4 and transglutaminase (TG) 2. IPF derived matrix increased expression of LOXL3 and TG2 transcripts, LOXL3 protein and TGase activity. Other cross linking enzymes were unchanged. To assess if IPF matrix affected fibroblast accumulation, I measured fibroblast adhesion, proliferation by MTT and EDU assays, and apoptosis by cleaved caspase 3, cleaved PARP and TUNEL assay on the different matrices. IPF matrix enhanced proliferation over control matrix in response to PDGF-BB. To determine if this pro-proliferative effect was dependent upon aberrant cross-linking we generated ECM from normal and IPF fibroblasts treated with cystamine dihydrochloride (TG2 inhibitor) or β-amino-proprionitrile (LOX family inhibitor). The enhanced fibroblast proliferation seen on IPF matrix was reduced close to levels of normal matrix by each cross link inhibitor. There was no effect on apoptosis induced by either FAS ligand or staurosporine when cells were seeded onto IPF or control matrix suggesting IPF ECM does not protect seeded fibroblasts from apoptosis. Bleomycin showed a trend towards increasing total lung hydroxyproline at day 24, 34 and 44 post administration however this was not statistically significant. Administration of cystamine at 40mg/kg/day showed no effect on total lung hydroxyproline. At day 34 post Bleomycin, cystamine administration showed a trend towards decreasing total lung hydroxyproline however again this was not statistically significant. Conclusions: The data supports the hypothesis that IPF derived matrix is structurally and functionally different from normal matrix. This results in enhanced fibroblast proliferation, adhesion and increased cross linking activity by effects on gene transcription. Inhibition of matrix cross-linking reduced this enhanced fibroblast adhesion and proliferation. Administration of cystamine dihydrochloride via IP injection for ten consecutive days at 100mg/kg/day in the Bleomycin model showed a trend towards decreasing total lung hydroxyproline.

Digitized by Kansas Correctional Industries

Airway pressure release ventilation (APRV) is a promising, unconventional mode of ventilation for the treatment of various respiratory diseases, including acute respiratory distress syndrome (ARDS). However, despite increasing global use and reported advantages of APRV over other modes, definitive conclusions cannot be drawn due to several concerns. First, the major confusion between APRV and other, similar modes, such as bi-phasic positive airway pressure (BIPAP). Second, clinical and methodological heterogeneity among published studies of APRV are understandably extensive, which contributes to outcome variability. Third, the absence of consensus on a standard protocol with clear rationale for the settings. This thesis provides an overview of the spectrum of ventilator settings that may be designated as APRV, summarises the research and clinical use status of APRV, exemplifies the need to clarify the characteristics that comprise the mode, and to assure reports of APRV use, from case reports through RCTs, including adequate data for a proper assessment. It encourages continued publication of observational as well as experimental clinical trial data, and discusses the feasibility of analysis strategies that may expand the information available from small patient samples. It also presents an unpublished, comprehensive, multifaceted clinical practice protocol (Al-ahmad protocol) for the use of APRV. Using the Al-ahmad protocol, four studies were conducted on non-spontaneously breathing patients who had ARDS (arising from a variety of pathologies). One study used a validated physiological simulator called integrated cardiopulmonary models (ICPMs) while the other three, were prospective cohort observational studies on real patients. The first study evaluated patients’ responses to changes in inspiratory pressure during conventional ventilation (CV) and APRV modes, using ICPMs vs. a real patient. The second study compared partial pressure of carbon dioxide (PaCO2) at any given ventilatory minute ventilation (MV) during CV and APRV. The third study aimed to identify proper configurations to optimise PaCO2 on patients with diverse pulmonary pathologies including restrictive (e.g. ARDS) and obstructive (e.g. chronic obstructive pulmonary disease) when using APRV. The fourth study compared oxygenation and haemodynamic status during CV and APRV. Results from ICPMs appeared to be analogous for both modes except for the significant difference in MV and tidal volume observed in the simulated vs. real APRV patients. We found in our clinical studies that compared to conventional modes, APRV was associated with significantly lower PaCO2 at significantly lower levels of MV, better oxygenation, and haemodynamic status.

Dead space is the part of tidal volume that does not participate in gas exchange and represents wasted ventilation. It is often increased in pulmonary diseases. Quantification of dead space by the original Bohr’s equation requires measuring mean alveolar pressure of CO2 (PACO2) and mixed expired partial pressure of CO2 (PĒCO2). Because of the difficulties and technical issues related with measuring PACO2 and PĒCO2, alternative methods have been proposed for the estimation of dead space. This thesis attempts to explore the performance of some methods proposed for the estimation of dead space to tidal volume ratio (VD/VT) in different pulmonary configurations and clinical scenarios. In the first study, we compared the performance of 5 different methods for the estimation VD/VT with the gold standard method in multiple ventilation/perfusion (V/Q) relationships. Six pulmonary configurations all with same alveolar dead space fraction of 0.25, but with different specific pattern of V/Q distribution were created within the Nottingham Physiology Simulator (NPS). Next, variations in the methods of estimating VD/VT upon varying 4 physiological factors were analysed. We concluded that the estimation of alveolar dead space ratio by 5 methods of estimating VD/VT is influenced by pattern of V/Q distribution and alterations in the relevant physiological factors. In the second study, we further analysed performance of 5 methods for estimating dead space ratio in patients with acute respiratory distress syndrome (ARDS). Nineteen ARDS subjects were created within the NPS and alveolar dead space fraction was measured by the gold standard method. Then, dead space fraction was determined by 5 different methods for estimating dead space fraction. We found that the estimates of dead space fraction measure different than the conventional equation in ARDS. In the third study, we compared efficacy of three lung recruitment maneuvers (RMs) in patients with ARDS. Six virtual ARDS patients were created and changes in dead space fraction, (Pa-E’CO2)/PaCO2 and other parameters were observed following the RMs. The results of this study showed that changes in (Pa-E’CO2)/PaCO2 closely relate with changes in VD/VT. These findings suggest that in clinical settings where it is not possible to measure dead space fraction, a simple estimate of VD/VT may be used to monitor the efficacy of RMs and titration of positive end-expiratory pressure. Simplified approaches for the estimation of dead space fraction may allow widespread use of this important physiological variable for diagnostic and prognostic purposes in critical care settings.