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The Single combination budesonide/formoterol inhaler as Maintenance And Reliever Therapy (SMART) regimen reduces severe asthma exacerbations, but it is uncertain whether it increases the risk of adverse effects due to high corticosteroid and beta-agonist doses with both short-term and cumulative exposure in patients at risk of severe exacerbations. The primary hypothesis was that the SMART regimen would reduce the risk of beta-agonist overuse. Secondary aims were to investigate whether patients treated with the SMART regimen were less likely to seek medical review in the setting of beta-agonist overuse and to determine whether any reduction in severe asthma exacerbations would be at a cost of a higher systemic corticosteroid burden. This 24-week, open-label, parallel-group, multicentre randomised controlled trial randomised 303 asthma patients with a recent exacerbation to combination 200/6µg budesonide/formoterol metered dose inhaler (MDI) according to the SMART regimen (two actuations twice daily as maintenance with one extra actuation as-needed for relief of symptoms) or a fixed-dose regimen (two actuations twice daily as maintenance) with one to two actuations of 100µg salbutamol MDI as-needed for relief of symptoms (the ‘Standard’ regimen), with electronic monitoring to measure actual medication use. The use of electronic monitoring allowed beta-agonist overuse to be applied as a marker of the risk of life-threatening asthma. The primary outcome was the proportion of participants with at least one high beta-agonist use episode (more than eight actuations per day of budesonide/formoterol in addition to the four maintenance doses in the SMART group or more than 16 actuations per day of salbutamol in the Standard group). There was no significant difference between groups in the proportion of participants with at least one high use episode: SMART 84/151 (55.6%) versus Standard 68/152 (44.7%), relative risk (95% CI) 1.24 (0.99 to 1.56), p=0.058. There were fewer days of high use in the SMART group [mean (SD) 5.1 days (14.3) versus 8.9 days (20.9), relative rate (95% CI) 0.58 (0.39 to 0.88), p=0.01]. Of the participants who had at least one high use episode, those in the SMART group had fewer days of high use without medical review [mean (SD) 8.5 days (17.8) versus 18.3 days (24.8), relative rate (95% CI) 0.49 (0.31 to 0.75), p=0.001]. The SMART regimen resulted in higher inhaled corticosteroid exposure [mean (SD) 943.5µg budesonide per day (1502.5) versus 684.3µg budesonide per day (390.5), ratio of means (95% CI) 1.22 (1.06 to 1.41), p=0.006], but reduced oral corticosteroid exposure [mean (SD) 77.5mg prednisone (240.5) versus 126.6mg prednisone (382.1), p=0.011], with no significant difference in composite systemic corticosteroid exposure [mean (SD) 793.7mg prednisone equivalent per year (893.1) versus 772.1mg prednisone equivalent per year (1062.7), ratio of means (95% CI) 1.03 (0.86 to 1.22), p=0.76]. Participants in the SMART group had fewer severe asthma exacerbations [35 (weighted mean rate per year 0.53) versus 66 (0.97), relative rate (95% CI) 0.54 (0.36 to 0.82), p=0.004]. The SMART regimen has a favourable risk/benefit profile in patients at risk of severe asthma exacerbations. Funding This study was funded by the Health Research Council of New Zealand, a government funding organisation.

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.

Rationale: Current asthma guidelines recommend reducing inhaled corticosteroid (ICS) therapy dose by 50% in patients with mild to moderate asthma who have demonstrated three months of good symptom control however there is evidence to suggest that this does not occur. Objectives: We tested whether exhaled nitric oxide (FeNO) measurements or other clinical indices could be utilised to predict a safe reduction of ICS dose, without provoking loss of symptom control or exacerbation within 3 months. We also investigated relationships between airway inflammation and asthma symptoms in the mild to moderate asthma cohort. Methods: 191 patients with stable asthma were recruited from primary care. Patients had their FeNO level measured at baseline and then had their inhaled corticosteroid (ICS) dose reduced by 50%. FeNO measurements were reassessed seven days later. The primary outcomes were whether baseline FeNO or a change in FeNO following ICS dose reduction could predict asthma stability at 3 months. Results: 128/191 patients (67%) completed the ICS dose reduction successfully at three months. 63/191 patients (33%) suffered from either a loss of control or an exacerbation. Baseline FeNO, or change in FeNO (post step-down minus pre step-down) were not statistically significantly different between the two groups. Conclusion: 67% of patients with well-controlled asthma can safely reduce their ICS dose by half without suffering from a loss of control or exacerbation within three months; however neither baseline nor change in FeNO measurements or routine clinical indices can be used to predict which patients can or cannot successfully tolerate a reduction in ICS dose.

Asthma is characterised by airway remodelling and an increase in airway resistance. A greater understanding of the mechanisms involved in airway inflammation and airway hyper-responsiveness (AHR) may highlight therapeutic opportunities for asthma. This study initially aimed to optimise the preparation of precision cut lung slices (PCLS) in mouse and pig to investigate the influence of calcium (Ca2+) homeostasis on airway smooth muscle (ASM) contraction as a prelude to human studies. The PCLS technique was then applied to a murine model of allergic airway disease to explore the inflammatory process and pathogenesis of airway hyper-reactivity in sensitised mice. Initial experiments using murine and porcine airways validated the PCLS model and demonstrated the significance of release and refilling of Ca2+ from internal stores to induce and maintain an airway contraction. Results also highlight interesting species differences in agonist sensitivity, with the porcine system sharing similar pharmacology to human airways. Using a murine model of allergic airway disease, agonist induced contractile responses in peripheral airways were measured in vitro using the PCLS technique. BALB/c mice underwent initial sensitisation by intraperitoneal administration of ovalbumin, receiving a 3 day challenge with aerosolised OVA l% (vlv), for varying periods of up to 3 weeks for acute, mid-chronic and chronic sensitisation protocols. To investigate the influence of the inflammatory environment, naive murine lung slices were incubated with selected inflammatory mediators. OVA sensitisation led to progressive structural remodelling and AHR to methacholine (MCh) challenge. However, this hyperresponsiveness was decreased 48 hours post lung removal. Of the inflammatory mediators selected for lung slice incubation, IL-33 significantly increased AHR to MCh. IL-33 is a proinflammatory cytokine with transcriptional repressor properties, playing a role in initiating the TH2 inflammatory response. In lung slices prepared from IL-33 receptor (ST2) KO mice IL-33 was unable to sensitise the contractile response. These data suggest the inflammatory environment promotes AHR and disassociates this airway sensitivity from structural remodelling. These data suggest a key role for IL-33 in mediating AHR in this murine model. Investigation of the mechanisms involved in airway hyper-reactivity revealed mRNA expression of IL-33 and the IL-33 receptor (ST2) in soluble and membrane bound forms were significantly increased in the mid-chronic and chronic ovalbumin sensitised murine lung tissue. Further quantitative analysis in human lung showed expression of IL-33 in epithelial and ASM cells. The human ST2 receptor (also known as IL-IRL-l) was expressed in mast cells. Together these results suggest IL-33 is a sensor of tissue damage; indirectly inducing AHR through further inflammatory cell activation to target ASM. This study demonstrates IL-33's role as an inflammatory marker of asthma and suggests a novel therapeutic intervention by targeting of the ST2 receptor.

Asthma is a heterogeneity disease that is mostly managed successfully using bronchodilators and anti-inflammatory drugs. Around 10%-15% of asthmatics however have difficult or severe asthma which is less responsive to treatments. Asthma and in particular severe asthma are now thought of a description of symptoms which may contain possible sub-groups with possible different pathologies which could be useful for targeting different drugs for different sub-groups. However little statistical work has been carried out to determine these sub-phenotypes. Studies have been carried out to partition severe asthma variables in to a number of sub-groups but the algorithms used in these studies are not based on statistical inference and it is difficult to select the number of best fitting sub-groups using such methods. It is also unclear where the clusters or sub-groups returned are actual sub-groups or reflect a bigger non-normal distribution. In the thesis we have developed a statistical model that combines factor analysis, a method used to obtain independent factors to describe processes allowing for variation over variables, and infinite mixture modelling, a process that involves determining the most probable number of mixtures or clusters thus allowing for variation over individuals. This model created is a Dirichlet process normal mixture latent variable model DPNMLVN and it is capable of determining the correct number of mixtures over each factor. The model was tested with simulations and used to analysis two severe asthma datasets and a cancer clinical trial. Sub-groups were found that reflect a high Eosinophilic group and an average eosinophilic group, a late onset older non atopic group and a highly atopic younger early onset group. In the clinical trial data 3 distinct mixtures were found relating to existing biomarkers not used in the mixture analysis.

Background: It is unclear how far the superior efficacy of omalizumab, established in randomized controlled clinical trials of patients with severe allergic asthma (SAA), translates into routine practice and when compared to matched controls. Methods: New-onset omalizumab-treated (OT) patients with SAA (n = 53) were compared to a matched control group of usual-care (UC) patients (n = 53). Treatment and procedures were naturalistic. Subsequent to a baseline assessment, patients were followed up over at least 6 months with at least two follow-up assessments. Primary clinical outcomes were the number of asthma attacks, persistence of asthma symptoms and degree of control [asthma control test (ACT), Global Initiative for Asthma]. Secondary outcome criteria were quality of life (Euro-Qol 5D) and number of medications. For each outcome we compared within-group effects from baseline to 6-month follow-up as well as between-group effects. Results: OT patients showed significant improvements in number [effect size (ES) = 0.03] and frequency (ES = 0.04) of asthma attacks as well as asthma control (ES = 0.09), whereas controls revealed no significant improvements in these measures. Further improvements in the OT group were found for ‘perceived control always’ (ACT, p = 0.006), no impairment (ACT, p = 0.02), reduction of sickness days (p = 0.002) and number of medications needed (p = 0.001). Conclusions: Substantial beneficial effects of omalizumab, similar to those observed in controlled trials and after marketing studies, were confirmed, particularly with regard to the reduction of asthma attacks, persistence of symptoms, asthma control and reduction of concomitant asthma medications. This study provides a tougher test and generalizable evidence for the effectiveness of omalizumab in routine care.

The evidence for genetic contributions to the development of asthma and atopy has been well established. Refining the major genetic factors underlying these contributions will lead to a greater understanding of the pathophysiology of these conditions and potentially identify novel therapeutic targets. This thesis describes a series of studies designed primarily using genome-wide association (GWA) approaches to examine common single nucleotide polymorphisms (SNPs) contributing to these phenotypes in the Caucasian population. Susceptibility to atopy was assessed using both non-parametric association tests of SNPs across the genome and focused analysis of two regions on chromosomes 3p22.1-q22.1 and 17p12-q24.3 previously identified through a meta-analysis of genome-wide linkage studies (GSMA). The discovery cohort consisted of 1,083 cases and 2,770 controls, replication analyses were undertaken in four independent population cohorts. A GWA study of severe asthma was carried out in 933 cases and 3,346 controls with replication in a further 231 cases and 1,345 controls. The contribution of SNPs within all previously reported asthma susceptibility loci identified using a comprehensive literature search was also evaluated. Overall, there is evidence for a large number of loci influencing both atopy and severe asthma, each harbouring modest effects. A number of potentially novel loci meeting nominal significance in both GWA studies have been identified requiring further work. Strong evidence was found to support the IL1RL1-IL33 signalling pathway in asthma pathogenesis. Molecular characterisation of the 5’ untranslated regions of IL1RL1 and IL33 suggest a complex regulatory role of identified common variants involving multiple promoters for IL1RL1. A number of asthma specific variants within the chromosome 2q12 and 9p24.1 regions were detected using next generation re-sequencing in homogenised pools of cases and controls warranting further analyses. This work has identified a potentially important pathway in which to focus the development of effective asthma therapies. Future directions will include functional analysis of replicated variants and tests of interactions between the multiple genetic and environmental factors likely to be involved in disease.