Chronic Obstructive Pulmonary Disease (COPD) presents an enormous public health challenge. Cigarette smoking remains the most important aetiological factor and although legislation to reduce smoking has been introduced in parts of the more developed world, consumption is increasing in many of the poorest parts of the world. With the predicted rise in disease prevalence, COPD is expected to become the worlds third largest cause of death by 2020. COPD is a disease state characterised by airflow limitation that is not fully reversible. Inhaled bronchodilators can only produce a small improvement in the airflow obstruction, but despite this, patients with COPD frequently use high doses of beta-2-agonists as the disease progresses and they develop breathlessness and exercise limitation. Short-acting beta-2-agonists are generally used as required to reduce breathlessness and reduce airflow obstruction whereas long-acting beta-2-agonists are prescribed on a regular twice-daily basis to reduce symptoms and rescue medication use and because of a potential beneficial effect on quality of life and exacerbation rates. Although generally well tolerated, the safety of inhaled beta-2-agonists has been a source of some concern since the late 1960s, when an epidemic of asthma deaths was associated with the use of a high dose formulation of isoprenaline. Further controversy has followed and questions have extended to long-acting beta-2-agonists, most notably after a recent large-scale post marketing surveillance study identified an association between the regular use of inhaled salmeterol and asthma-related deaths. The safety of inhaled beta-2-agonists is also an important consideration for patients with COPD. Being older and likely to have a history of cigarette consumption means that they are at risk of having symptomatic, or subclinical, ischaemic heart disease. Beta-2-agonists cause a number of systemic effects including an increase in heart rate, transient hypoxaemia and hypokalaemia. Since many patients with COPD are already hypoxaemic and may be taking other drugs that stimulate the myocardium and cause hypokalaemia, the additional systemic effects from beta-2-agonists may be more likely to produce adverse cardiac events including dysrhythmia and ischaemia. This thesis is concerned with the safety of inhaled beta-2-agonists in the management of COPD. The introduction consists of an overview of the epidemiology, natural history and pathology of COPD (Chapter 1) and a review of human beta-2-adrenoceptor function and inhaled beta-2-agonist pharmacology (Chapter 2). This is followed by a systematic literature review of the results from long-term clinical studies of inhaled beta-2-agonists in subjects with COPD (Chapter 3). The original work consists of three clinical studies that have examined aspects of the effect of high dose inhaled beta-2-agonists in subjects with COPD and a discussion to place these findings in context. Most published studies of inhaled beta-2-agonists in subjects with COPD have focused on their efficacy, rather than safety. We were concerned that some individuals with COPD and limited bronchodilator reversibility may experience an increase in adverse systemic effects after inhaling high doses of beta-2-agonists, which could lead to detrimental outcomes in certain clinical situations. Apart from the cardiac effects mentioned above, beta-2-agonists increase tremor, which causes CO2 production, and cardiac output and tissue perfuson, which increases the transport of CO2 to the lungs. The increase in CO2 flux to the lungs will normally increase ventilation. We were concerned however that some subjects with severe COPD would not be able to increase ventilation appropriately in response to the beta-2-agonist and this would lead to an increase in PaCO2. Our hypothesis was that high dose inhaled beta-2-agonists could worsen respiratory failure in some subjects with severe COPD. The first two studies in the thesis examined the effect of high dose inhaled salbutamol on the partial pressure of arterial oxygen and carbon dioxide in subjects with severe COPD. We initially conducted a double blind, randomised study on subjects within 48 hours of being admitted to hospital with an acute exacerbation of COPD (Chapter 4). The study was designed to determine whether high dose salbutamol caused an increase in the partial pressure of arterial carbon dioxide. We randomised subjects at a ratio of 3:1 to receive either salbutamol or ipratropium bromide and studied the pharmacodynamic effect on heart rate, PaO2 and PaCO2 over five hours. Over eighteen months and despite extensive efforts I was only able to recruit ten subjects, of whom five completed the study. I found that subjects who required hospital admission with an acute exacerbation of COPD were either too unwell for the study, had co-morbidities that precluded participation or the individuals were unwilling to participate. Although the study was terminated prematurely and we were unable to perform statistical analysis, I have presented the findings from the five subjects who completed the study, of whom four were randomised to receive salbutamol. We used ascending doses of salbutamol (1.25mg, 1.25mg, 25mg, 5mg, 5mg) and found no consistent effect on PaCO2 or PaO2 and no dose response relationship. The subject with the highest baseline PaCO2 did however have a rise in PaCO2 with the highest 5mg doses of salbutamol. To test the hypothesis further we conducted a randomised, double blind, crossover study and examined the effect of salbutamol on the arterial blood gas tensions of fourteen patients with stable severe COPD and a history of chronic or intermittent hypercapnia. The study was designed to determine whether high dose salbutamol causes a rise in PaCO2 when inhaled by subjects with severe COPD and a history of alveolar hypoventilation. We compared the effect of two 5mg doses with two 200 microgram doses of salbutamol on PaO2 and PaCO2 and heart rate. The subjects had severe COPD with a mean FEV1 of 0.71 L (27% predicted) and a mean smoking history of 53 pack years. The mean baseline PaO2 was 7.9 kPa and the mean baseline PaCO2 was 7.0 kPa. The high dose of salbutamol caused a mean fall in both PaO2 and PaCO2 and a small increase in heart rate. There was some support for our hypothesis however as three subjects had a small rise in PaCO2 after high dose nebulised salbutamol (Chapter 5). The third study was a double blind, crossover, dose-response examination of the bronchodilator and systemic effects of inhaled formoterol in subjects with COPD (Chapter 6). The rapid onset and prolonged duration of action of formoterol offers potential for the drug to be used as rescue medication in addition to twice daily maintenance therapy, as is the case in the management of asthma. Our hypothesis was that high doses of formoterol would produce adverse systemic effects that would outweigh the beneficial bronchodilator effects in subjects with COPD and limited bronchodilator response to salbutamol. We studied 20 subjects, with a mean FEV1 of 1.32 L (47% predicted) and a mean smoking history of 42 pack years. Each subject was studied on five days and after receiving placebo, formoterol 6, 12, 24 and 48 mg in a random sequence, we examined the effect of each dose on FEV1, tremor, dyspnoea, heart rate, blood pressure, SpO2, walk distance, potassium and satisfaction. We found that all doses were well tolerated and although there was a small dose related increase in FEV1 and the mean satisfaction scores were higher with each dose of formoterol than placebo, there was no dose related improvement in measures that are important to the patient, including breathlessness and walk distance. Apart from a dose related increase in tremor, other systemic effects were limited. All three studies found that high dose inhaled beta-2-agonists produced relatively modest systemic effects in subjects with COPD.

This thesis used a qualititave research approach to explore the causes, manifestations and consequences of tuberculosis (TB) stigma in an urban district in Ghana. It examined reasons why TB is stigmatised and elucidated how TB stigma manifests within the community setting and the healthcare system. It also explored the feelings and experiences of TB patients, to highlight how the fear of stigmatisation may affect case finding and treatment adherence. Twenty eight focus groups (6 with patients, 6 with health workers and 16 with community members) and 121 individual interviews (66 with community members, 34 with patients and 21 with health staff) were conducted. Data were analysed using Grounded Theory techniques and procedures. Eleven causes of TB stigma were identified: fear of infection; physical frailty of TB patients; association of TB with HIV/AIDS; perceived causes and spread of TB; outdated societal practices about TB; public health practice and discourse; attitudes of healthcare workers towards TB patients; health staff’s own fear of TB; self-stigmatisation by TB patients; judgement, blaming and shaming TB patients; and past experiences with TB. Elements of physical and moral threats were identified in all these causes of TB stigma. The threat the disease poses to community members led to imposition of socio-physical distance, participatory restrictions and rules for unexpected interactions on those suffering from TB in society. Within the healthcare system, the threat of TB affected the attitudes and behaviours of healthcare workers towards TB patients and TB work. Health managers also sited TB units/wards in isolated parts of the hospital, and failed to provide adequate tools and equipment, support and supervision to enable the provision of quality TB services. The fear of stigmatisation made the patients deny the obvious symptoms of the disease, and report to the hospital only after prolonged period of self-medication in the community. When diagnosed, they cried, questioned how they got the disease, contemplated committing suicide and were mostly isolated within the family and community. For everyone, the threat of TB underlies their beliefs, attitudes, actions and behaviours when interacting with TB patients. It also forms the basis of avoidance of social interactions, and attitudes and practices of healthcare workers towards TB patients. The TB control programme should encourage open discussion about TB in the community and tailor health education messages to the community’s understanding of the disease. TB services should be completely integrated into the general healthcare system and community members involved in activities of the TB control programme. Regular refresher courses in TB control and management should be organised for health professionals and a national guideline for the prevention of TB in health workers developed.

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 This thesis investigates the effect of sodium modification on asthma and heart rate variability (HRV) using a randomised controlled study design. Cross-sectional data were used to assess novel markers of asthma control and if serum osmolality was inversely associated with lung function. Methods and Results The effect of dietary sodium modification on asthma control and HRV The study design was a randomised placebo-controlled double-blind study of participants aged 18-65 years old with asthma and measurable bronchial reactivity. The intervention involved adopting a low sodium diet for six weeks. 199 participants provided measures of asthma control and 29 participants provided 24-hour electrocardiographic measurements at baseline and six weeks later. There were no differences between those allocated to the low and normal sodium diet groups in any of the measures of asthma control or HRV. Bronchoconstriction induced by deep inspiration and exhaled carbon monoxide as surrogate marker of asthma control Serial PEFR measures were provided by 127 individuals to measure bronchoconstriction after deep inhalation and 182 individuals provided measurements of eCO. The relationship between these measures and asthma control was assessed. There was no relationship between either bronchoconstriction after deep inspiration or eCO and asthma control. Serum osmolality and lung function The NHANES III database was used to investigate if serum osmolality was cross-sectionally associated with lung function. In 13,602 individuals a SD increase in serum osmolality was associated with a decrease in FEV1 of 19.8ml (95% CI; -30.3 to -9.3) and FVC of 35.3ml (95% CI; -47.9 to -22.7). Conclusions A low sodium diet did not improve asthma control or increase heart rate variability in adults with asthma. Bronchoconstriction to deep inspiration and measurements of eCO have limited potential as markers of asthma control. In the NHANES dataset, increased serum osmolality was associated with decreased lung function.

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.

No description available.

jdelvall@upc.edu.pe / Acute respiratory infections are responsible for high morbi–mortality in Peruvian children. However, the etiological agents are poorly identified. This study, conducted during the pandemic outbreak of H1N1 influenza in 2009, aims to determine the main etiological agents responsible for acute respiratory infections in children from Lima, Peru. Nasopharyngeal swabs collected from 717 children with acute respiratory infections between January 2009 and December 2010 were analyzed by multiplex RT-PCR for 13 respiratory viruses: influenza A, B, and C virus; parainfluenza virus (PIV) 1, 2, 3, and 4; and human respiratory syncytial virus (RSV) A and B, among others. Samples were also tested with direct fluorescent-antibodies (DFA) for six respiratory viruses. RT-PCR and DFA detected respiratory viruses in 240 (33.5%) and 85 (11.9%) cases, respectively. The most common etiological agents were RSV-A (15.3%), followed by influenza A (4.6%), PIV-1 (3.6%), and PIV-2 (1.8%). The viruses identified by DFA corresponded to RSV (5.9%) and influenza A (1.8%). Therefore, respiratory syncytial viruses (RSV) were found to be the most common etiology of acute respiratory infections. The authors suggest that active surveillance be conducted to identify the causative agents and improve clinical management, especially in the context of possible circulation of pandemic viruses