Background The rate at which the prevalence of allergic disease is increasing in many countries suggests that environmental exposures may be important aetiological factors. Epidemiological evidence indicates that infection with helminth parasites may be one such factor: in particular, in a systematic review and meta-analysis, current hookworm (Necator americanus) infection at an intensity of 50 eggs/g faeces was shown to be associated with a halving of risk of asthma. The relation between parasite infection and atopy has not been subjected to the same rigorous and comprehensive review. Based on the results of the studies in asthma, it is possible that hookworm infection may have potential in the treatment of this disease, but to date, no clinical trials have been carried out to test this hypothesis. For ethical and safety reasons, before embarking on a clinical trial in asthma it is necessary to establish the dose of larvae required to produce at least 50 eggs/g faeces, and to determine whether experimental hookworm infection might exacerbate bronchial hyper-responsiveness during larval lung migration. Aims and objectives The first aim of this thesis was to establish whether experimental hookworm infection improves asthma by carrying out a series of three intervention studies. The second aim was to determine the association between intestinal parasite infection and atopy (defined as positive allergen skin sensitisation or the presence of specific IgE) and to establish whether the association was species-specific. This thesis therefore consists of two main components: a series of three clinical trials of experimental hookworm infection; and a systematic review and metaanalysis of the association between intestinal parasite infection and atopy. Methods and Results Dose-ranging study of experimental hookworm infection Aim: To identify the dose of hookworm larvae necessary to achieve 50 eggs/g faeces and to monitor any adverse effects of infection. Methods: Ten healthy volunteers, without asthma or bronchial responsiveness to inhaled methacholine, received 10, 25, 50, or 100 Necator americanus larvae administered double-blind to an area of skin on the arm and were monitored weekly for 12 weeks. Results: All doses resulted in the production of at least 50 eggs/g faeces in the eight subjects who completed the study. Skin itching at the entry site and gastrointestinal symptoms were common at higher doses. Study of experimental hookworm infection in allergic rhinoconjunctivitis Aim: To determine whether hookworm larval migration through the lungs increases bronchial responsiveness in allergic individuals with measurable bronchial responsiveness but not clinical asthma, and to investigate the general tolerability of infection and its effect on allergic symptoms. Methods: Thirty individuals with allergic rhinoconjunctivitis and measurable bronchial responsiveness to adenosine monophosphate (AMP) but not clinically diagnosed asthma were randomised, double-blind, to cutaneous administration of either ten Necator americanus larvae or histamine placebo, and followed for 12 weeks. The primary outcome was the maximum fall from baseline in provocative dose of inhaled AMP required to reduce one-second forced expiratory volume by 10% (PD10AMP) measured at any time over the four weeks after active or placebo infection. Secondary outcomes included peak flow variability in the four weeks after infection, adverse effect diary scores and rhinoconjunctivitis symptom severity over the 12-week study period, and change in allergen skin sensitisation between baseline and 12 weeks. Results: Mean maximum change in PD10AMP from baseline was slightly but not significantly greater in the hookworm than the placebo group (-1.67 and -1.16 doubling doses; mean difference -0.51, 95% confidence interval: -1.80 to 0.78; p=0.42). There were no significant differences in peak flow variability, rhinoconjunctivitis symptoms or allergen skin sensitisation between groups. Symptom scores of potential adverse effects were more commonly reported in the hookworm group, but infection was generally well tolerated. Study of experimental hookworm infection in asthma Aim: To determine the effects of experimental hookworm infection on asthma. Methods: Thirty-two individuals with asthma and measurable bronchial hyperresponsiveness to adenosine monophosphate (AMP) were randomised, doubleblind, to cutaneous administration of either ten Necator americanus larvae or histamine placebo, and followed for 16 weeks. The primary outcome was the change in provocation dose of inhaled AMP required to reduce one-second forced expiratory volume by 20% (PD20AMP) from baseline to week 16. Secondary outcomes included change in several measures of asthma control and allergen skin sensitisation and the occurrence of adverse effects. Results: Mean PD20AMP improved in both groups, more in the hookworm (1.49 doubling doses (DD)) than the placebo group (0.98 DD), but the difference between groups was not significant (0.51 DD, 95% confidence interval: -1.79 to 2.80; p=0.65). There were no significant differences between the two groups for other measures of asthma control or allergen skin sensitisation. Infection was generally well tolerated. Systematic review and meta-analysis of the association between intestinal parasite infection and atopy Aim: To quantify the association between current intestinal parasite infection and the presence of atopy in a systematic review and meta-analysis of epidemiological studies, and to determine whether, as with asthma, this relation is species-specific. Methods: MEDLINE, EMBASE, LILIACS and CAB Abstracts (to March 2009); reviews; and reference lists from publications were searched. No language restrictions were applied. Studies that measured current parasite infection using direct faecal microscopy and defined atopy as allergen skin sensitisation or presence of specific IgE were included. Pooled odds ratios (OR) and 95% confidence intervals (95% CI) using data extracted from published papers using random effect models were calculated. Results: 20 studies met the inclusion criteria. Current parasite infection was associated with a reduced risk of allergen skin sensitisation (OR 0.69, 95% CI: 0.60 to 0.79; p<0.01). When analyses were restricted to current geohelminth infection, the size of effect remained similar (OR 0.68, 95% CI: 0.60 to 0.76; p<0.01). In species-specific analysis, a consistent protective effect was found for infection with Ascaris lumbricoides, Trichuris trichiura and hookworm. There were insufficient data to pool results for chistosomiasis or atopy defined by presence of specific IgE. Conclusions Experimental infection with ten Necator americanus larvae produces at least 50 eggs/g faeces, the intensity of infection seen to protect against asthma in observational studies. This dose is safe, well tolerated, feasible to use in clinical trials and does not cause clinically significant exacerbation of bronchial responsiveness during larval pulmonary migration. In clinical trials, it did not result in significant improvement in symptoms of allergic rhinoconjunctivitis, or in bronchial hyper-responsiveness or other measures of asthma control. However, a non-significant improvement in bronchial hyper- responsiveness was seen, indicating that further studies incorporating revised dosing regimens that more closely mimic natural infection are feasible, and should be undertaken, with the aim of identifying novel treatments for asthma. As with asthma, there appears to be an inverse association between intestinal parasite infection and atopy. Work should continue to identify the mechanisms of this effect and means of harnessing these to reduce the global burden of allergic disease.

The urokinase plasminogen activator receptor (PLAUR) is a membrane anchored receptor that has been associated with a number of disease states. In these diseases, elevated receptor levels were associated with increased disease aggressiveness and higher mortality rates. Through genetic studies PLAUR has been identified as an asthma susceptibility gene. In these studies, coding and untranslated region single nucleotide polymorphisms showed association with asthma diagnosis and decline in lung function. In addition, association with baseline lung function in smokers and PLAUR SNPs was identified. This suggests that PLAUR plays a role in respiratory disease. Work presented in this thesis aimed to i) identify whether serum levels of PLAUR are associated with obstructive lung disease and lung function parameters, ii) identify novel regulatory mechanisms determining PLAUR levels, both at the genetic level in primary bronchial epithelial cells and at the protein level for serum PLAUR, iii) explore a role for the different isoforms in asthma and COPD and iv) determine novel variation in the PLAUR gene and 5` and 3` distal regions through next generation sequencing. Levels of the soluble cleaved form of PLAUR in serum were determined to be significantly elevated in COPD and asthma subjects compared to a control population. This identified an association between the soluble cleaved receptor and disease per se. However, PLAUR levels in serum could not be related to lung function parameters. With regards to receptor regulation, a genome-wide association study identified a novel post-translational PLAUR regulatory mechanism. This involved key SNPs in the human plasma kallikrein gene promoter that directed human plasma kallikrein enzymatic activity to cleave PLAUR in a post-translational mechanism. PLAUR gene regulation was also investigated via molecular biology, identifying that in primary bronchial epithelial cells, PLAUR regulation involves the gene’s 5 prime and 3 prime untranslated regions. Investigation of regulation under multiple stimulations pertinent to respiratory disease identified that cigarette smoke extract selectively elevated the soluble spliced variant of the receptor through a three prime untranslated region mechanism. This suggests that bronchial epithelial damage driven by cigarette smoke may be at least partially mediated by the soluble spliced form of PLAUR. Overexpression of PLAUR identified that the receptor has an important role in regulating primary bronchial epithelial cell function, including migration and rate of mitochondrial activity. Interestingly, results identified isoform specific roles for the different forms of the receptor suggesting that variant-specific over-expression of PLAUR could have diverse effects on cell function. Importantly this study was also the first to define a role for the soluble spliced form of PLAUR. Investigation into variation in the PLAUR gene and surrounding regions through next generation sequencing in asthma (n=200) and control (n=200) populations identified a number of novel variants including 4 variants unique to asthma population. In summary, the work described in this thesis has identified a novel association between serum soluble cleaved PLAUR and obstructive lung disease, as well defining novel genetic and post-translational regulatory mechanisms for PLAUR, importantly defining isoform specific PLAUR regulation for the first time. This work has also identified novel isoform specific roles for PLAUR, which have significant modulatory effects on bronchial epithelial cell function, and has through next generation sequencing furthered knowledge on universal and asthma specific PLAUR variation.

Bronchiolitis is the commonest cause of acute respiratory failure in infancy and several hundred children need respiratory support for the condition each year in the United Kingdom. Continuous negative extrathoracic pressure (CNEP) has been used to support such children but concerns about its possible association with significant harm prompted a government enquiry into the conduct of research at a UK centre using the technique. This retrospective study was designed to address these concerns by careful evaluation of outcome in two matched cohorts. Fifty children who had received CNEP for bronchiolitis as infants were compared with 50 controls who were treated in another hospital during the same period. Pre-treatment variables, demographics and neonatal factors were well matched in the two groups. In all subjects questionnaires and clinical examination were used to assess respiratory symptoms, disability and health-related quality of life whilst respiratory function was assessed by measuring airway resistance using the interrupter technique (Rint), by spirometry and by bronchodilator responsiveness. CNEP was associated with reduced need for, and shorter duration of, positive pressure ventilation but with longer periods in oxygen and hospital. Median Rint was 16.5% higher in the CNEP cohort (p<0.001) and median FEF25-75 was 9.3% lower (p=0.029). There were no significant differences between the groups in FEV1, FVC, bronchodilator responses or respiratory symptoms, or in the prevalence of moderate or severe disability (Mantel-Haenszel statistic 1.40, 95% confidence intervals: 0.64 -3.04, p=0.39). Median health utility indices were similar; CNEP 1.00 (interquartile range: 0.85-1.00), controls 0.99 (interquartile range: 0.81 -1.00), n=48 pairs, p= 0.37. The higher Rint and lower FEF25-75 in the CNEP group represent a small difference in respiratory function that may be attributable to population differences but a CNEP effect cannot be excluded. Further evaluation of the use of CNEP in bronchiolitis requires a prospective, controlled study.

According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) Management Guidelines (2001), the definition of COPD is “a disease state characterised by airflow limitation that is not fully reversible. The airflow is usually progressive and associated with inflammatory responses of the lungs to noxious particles and gases.” It is becoming an increasing prevalent problem worldwide, with the incidences of morbidity and mortality continually increasing and promoting a lower quality of life in individuals that continue to suffer from it. To date, there is still an incomplete understanding of the pathogenesis of the disease resulting in poor diagnosis and treatment plans for COPD that are insufficient in preventing a decline in lung function. In recent years, research has focussed on discovering a set of biomarkers that could improve our understanding of pathogenesis of disease. The ability to measure a vast array of biomarkers simultaneously is highly desirable however the cost associated is somewhat prohibitive. Current methods centre on measuring the presence or absence of multiple biomarkers in patient samples compared to controls. As COPD is a multi-component disease which encompasses diseases such as emphysema and chronic bronchitis, it may be necessary to look at biomarker patterns within each disease category. A variety of immune effector cells are known to lead to the pathophysiology of COPD including neutrophils, macrophages and CD8 T-lymphocytes that are all documented to be increased in number and contribute to the inflammatory process. Protein microarrays are used as a measurement tool to determine and quantify the presence or amount of proteins that exist in biological samples (i.e. blood, sputum, [iii] urine etc). The wide use of protein microarray technology has advanced diagnosis and management of multifactorial diseases such as cancer, autoimmunity and allergy. At present, multiple microarray kits are available to researchers at a large cost which make it impractical for most research groups to investigate multiple biomarkers of interest simultaneously. Here we show development, validation and implementation of our bespoke in-house microarray platform enabling quantitative and simultaneous analysis of multiple protein biomarkers at a reasonable cost. The methodology is based on the traditional sandwich ELISA; antibodies are immobilised on poly-L-lysine coated glass and signals amplified and quantified through fluorescence. The accuracy and reproducibility of the in-house microarray was investigated using the guidelines outlined by the Food and Drug Administration (FDA) for pharmacokinetic assay validation. The assay was shown to have high reproducibility with assay accuracy between 80-120% and precision within 20% coefficient of variation, except in very low abundant cytokines such as IL-10, where the CVs were higher due to the variation at the lowest concentrations in sera. Importantly there were no significant differences between ELISA and microarray. This microarray platform was then used to study a selection of healthy controls (n=12), healthy smoking controls (n=36) and COPD patients (n=60) to see if there was a difference in the expression of the 16 biomarkers tested. The overall analysis of the 16 biomarkers investigated in this study, a significant increase in expression of eotaxin-2 was observed in the sera those that have COPD compared to healthy controls and healthy smoking controls. This suggests that eoxtaxin-2 may potentially be responsible for the recruitment and activation of multiple cytokines which in turn lead to the inflammatory cascade observed in COPD COPD severity is divided into four categories according to international guidelines outlined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD). This is often known as stage 1 (mild), stage 2 (moderate), stage 3 (severe) and stage 4 (very severe). This is based on the forced expiratory volume per second (FEV-1%). Interestingly when investigating the different severities of GOLD in COPD, it was observed that at the highest stage of GOLD (stage 4), the expression of 15 of the 16 biomarkers had dropped significantly in comparison to the other stages. This may suggest that at this point of the disease process, the immune system may in fact be suppressed in alliance to hypoxia experienced by an individual. Additionally it has to be acknowledged that the medication that the COPD patients were on were not available prior to analysis. It has to be taken into account that patients at GOLD stages 3 and 4 could be likely to be on a high dose of inhaled corticosteroids, which are immunosuppressive which would lead to drop in the 15 cytokines observed. However without the information available, it cannot be definitive to make such conclusions. Hence this work offers an understanding into the development of a bespoke microarray platform that is capable of investigating protein biomarkers in any disease setting.

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.

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.

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.