Eh Walters

Airway inflammation, basement membrane thickening and bronchial hyperresponsiveness in asthma

Background: There are few data in asthma relating airway physiology, inflammation and remodelling and the relative effects of inhaled corticosteroid (ICS) treatment on these parameters. A study of the relationships between spirometric indices, airway inflammation, airway remodelling, and bronchial hyperreactivity (BHR) before and after treatment with high dose inhaled fluticasone propionate (FP 750 μg bd) was performed in a group of patients with relatively mild but symptomatic asthma. Methods: A double blind, randomised, placebo controlled, parallel group study of inhaled FP was performed in 35 asthmatic patients. Bronchoalveolar lavage (BAL) and airway biopsy studies were carried out at baseline and after 3 and 12 months of treatment. Twenty two normal healthy non-asthmatic subjects acted as controls. Results: BAL fluid eosinophils, mast cells, and epithelial cells were significantly higher in asthmatic patients than in controls at baseline (p<0.01). Subepithelial reticular basement membrane (rbm) thickness was variable, but overall was increased in asthmatic patients compared with controls (p<0.01). Multiple regression analysis explained 40% of the variability in BHR, 21% related to rbm thickness, 11% to BAL epithelial cells, and 8% to BAL eosinophils. The longitudinal data corroborated the cross sectional model. Forced expiratory volume in 1 second improved after 3 months of treatment with FP with no further improvement at 12 months. PD20 improved throughout the study. BAL inflammatory cells decreased following 3 months of treatment with no further improvement at 12 months (p<0.05 v placebo). Rbm thickness decreased in the FP group, but only after 12 months of treatment (mean change –1.9, 95% CI –3 to –0.7 μm; p<0.01 v baseline, p<0.05 v placebo). A third of the improvement in BHR with FP was associated with early changes in inflammation, but the more progressive and larger improvement was associated with the later improvement in airway remodelling. Conclusion: Physiology, airway inflammation and remodelling in asthma are interrelated and improve with ICS. Changes are not temporally concordant, with prolonged treatment necessary for maximal benefit in remodelling and PD20. Determining the appropriate dose of inhaled steroids only by reference to symptoms and lung function, as specified in current international guidelines, and even against indices of inflammation may be over simplistic. The results of this study support the need for early and long term intervention with ICS, even in patients with relatively mild asthma.

beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels.

Background: The use of reverse transcription-polymerase chain reaction (RT-PCR) to measure mRNA levels has led to the common use of β-actin and GAPDH housekeeping genes as denominators for comparison of samples. Expression of these genes is assumed to remain constant, so normalising for variations in processing and signal quantitation. However, it is well documented that β-actin and GAPDH expression is upregulated with proliferation, activation, and differentiation. We hypothesised that airway samples which differ in their cellular profiles and activation status have different levels of expression of GAPDH and β-actin. Methods: The mRNA for β-actin, GAPDH, and interleukin (IL)-2 was measured in bronchoalveolar lavage (BAL) fluid cells and endobronchial biopsy tissue by competitive RT-PCR in a cross sectional study of 26 normal controls and 92 asthmatic subjects. Results: For both BAL fluid cells and biopsy tissue, asthmatics overall had reduced expression of GAPDH and β-actin mRNA. In asthmatic subjects not using inhaled corticosteroids (ICS), GAPDH mRNA levels in both BAL fluid and biopsy tissue, and β-actin mRNA in BAL fluid cells were 10 times lower than samples from both normal controls and from asthmatic subjects using ICS. β-Actin mRNA in biopsy specimens showed the same pattern of expression, but asthmatic subjects not using ICS were not significantly different from those receiving ICS treatment. IL-2 mRNA levels did not differ between the subject or treatment groups but, when expressed as a ratio with β-actin, significant differences were seen. Conclusions: β-Actin and GAPDH used as denominators of gene expression quantitation in asthma research can cause confounding. Housekeeping genes need careful validation before their use in such quantitative mRNA assays.

Effect of high flow oxygen on mortality in chronic obstructive pulmonary disease patients in prehospital setting: randomised controlled trial

Objectives To compare standard high flow oxygen treatment with titrated oxygen treatment for patients with an acute exacerbation of chronic obstructive pulmonary disease in the prehospital setting. Design Cluster randomised controlled parallel group trial. Setting Ambulance service in Hobart, Tasmania, Australia. Participants 405 patients with a presumed acute exacerbation of chronic obstructive pulmonary disease who were treated by paramedics, transported, and admitted to the Royal Hobart Hospital during the trial period; 214 had a diagnosis of chronic obstructive pulmonary disease confirmed by lung function tests in the previous five years. Interventions High flow oxygen treatment compared with titrated oxygen treatment in the prehospital (ambulance/paramedic) setting. Main outcome measure Prehospital or in-hospital mortality. Results In an intention to treat analysis, the risk of death was significantly lower in the titrated oxygen arm compared with the high flow oxygen arm for all patients (high flow oxygen n=226; titrated oxygen n=179) and for the subgroup of patients with confirmed chronic obstructive pulmonary disease (high flow n=117; titrated n=97). Overall mortality was 9% (21 deaths) in the high flow oxygen arm compared with 4% (7 deaths) in the titrated oxygen arm; mortality in the subgroup with confirmed chronic obstructive pulmonary disease was 9% (11 deaths) in the high flow arm compared with 2% (2 deaths) in the titrated oxygen arm. Titrated oxygen treatment reduced mortality compared with high flow oxygen by 58% for all patients (relative risk 0.42, 95% confidence interval 0.20 to 0.89; P=0.02) and by 78% for the patients with confirmed chronic obstructive pulmonary disease (0.22, 0.05 to 0.91; P=0.04). Patients with chronic obstructive pulmonary disease who received titrated oxygen according to the protocol were significantly less likely to have respiratory acidosis (mean difference in pH 0.12 (SE 0.05); P=0.01; n=28) or hypercapnia (mean difference in arterial carbon dioxide pressure −33.6 (16.3) mm Hg; P=0.02; n=29) than were patients who received high flow oxygen. Conclusions Titrated oxygen treatment significantly reduced mortality, hypercapnia, and respiratory acidosis compared with high flow oxygen in acute exacerbations of chronic obstructive pulmonary disease. These results provide strong evidence to recommend the routine use of titrated oxygen treatment in patients with breathlessness and a history or clinical likelihood of chronic obstructive pulmonary disease in the prehospital setting. Trial registration Australian New Zealand Clinical Trials Register ACTRN12609000236291.

Biological dust exposure in the workplace is a risk factor for chronic obstructive pulmonary disease

Background: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality. Although the main risk factor is smoking, 15–19% of COPD even in smokers has been attributed to occupational exposures. The aim of this study was to investigate the association between occupational exposure and risk of COPD. Methods: Participants were part of a cross sectional study of risk factors for COPD. A total of 1232 completed a detailed respiratory questionnaire, spirometric testsing and measurement of gas transfer. Job histories were coded according to the International Standard Classification of Occupations. These codes were then used to establish occupational exposures using the ALOHA job exposure matrix. Results: The prevalence of emphysema was 2.4%, chronic obstructive bronchitis 1.8%, and COPD 3.4%. Subjects ever exposed to biological dusts had an increased risk of chronic obstructive bronchitis (OR 3.19; 95% CI 1.27 to 7.97), emphysema (OR 3.18; 95% CI 1.41 to 7.13), and COPD (OR 2.70, 95% CI 1.39 to 5.23). These risks were higher in women than in men. For biological dust, the risk of emphysema and COPD was also significantly increased in both the duration of exposure categories, again in women but not in men. No significant increased risks for COPD were found for mineral dust (OR 1.13; 95% CI 0.57 to 2.27) or gases/fumes (OR 1.63; 95% CI 0.83 to 3.22). Conclusion: In this general population sample of adults, occupational exposures to biological dusts were associated with an increased risk of COPD which was higher in women. Preventive strategies should be aimed at reducing exposure to these agents in the workplace.

Number and activity of inflammatory cells in bronchoalveolar lavage fluid in asthma and their relation to airway responsiveness.

Bronchial responsiveness to inhaled methacholine was measured four to six days before fibreoptic bronchoscopy in 22 asthmatic patients (10 smokers) and 20 control subjects (12 smokers). The asthmatic patients had a baseline FEV1 greater than 60% predicted and a PD20FEV1 (provocative dose of methacholine causing a 20% fall in FEV1) of 0.006-3.7 mg. The 20 control subjects had normal pulmonary function and a PD20FEV1 above the maximum cumulative dose of methacholine of 6.4 mg. Bronchoalveolar lavage of a middle lobe segment (lingula in four subjects) was performed with three sequential 60 ml aliquots of sterile saline. Cellular metabolic activity was stimulated with latex in aliquots of resuspended cells, and measured by means of luminol enhanced chemiluminescence to assess neutrophil activity and lucigenin enhanced chemiluminescence to assess macrophage activity. Mean absolute total cell counts were similar in the asthmatic and control groups but there were differences in differential cell counts, with a significant increase in eosinophil (p less than 0.05) and lymphocyte (p less than 0.005) counts in asthma. PD20FEV1 was negatively correlated with percentage neutrophil counts (p less than 0.005). Luminol enhanced chemiluminescence/1000 neutrophils was increased about twofold in asthmatic subjects (p less than 0.001), but was not correlated with PD20FEV1 Lucigenin enhanced chemiluminescence/1000 macrophages was increased nearly fourfold in asthmatic patients (p less than 0.001) and showed a negative correlation with PD20FEV1 (p less than 0.01). The macrophage count was increased twofold in current smokers in both groups, but other cell numbers were not altered significantly. Smoking did not affect cellular metabolic activity in either group. This study supports the idea that an inflammatory process is present in the airways of those with asthma, and suggests a relation between bronchial responsiveness and both neutrophil numbers and macrophage activity.

International prevalences of reported food allergies and intolerances. Comparisons arising from the European Community Respiratory Health Survey (ECRHS) 1991-1994

Objective: The aim of this study was to report the prevalence, type and reported symptoms associated with food intolerance.Design: A cross-sectional epidemiological study involving 15 countries using standardized methodology. Participants answered a detailed interviewer-administered questionnaire and took part in blood, lung function and skin prick tests to common aeroallergens.Setting: Randomly selected adults who took part in the second phase of the European Community Respiratory Health Survey (ECRHS).Subjects: The subjects were 17280 adults aged 20–44 y.Results: Twelve percent of respondents reported food allergy/intolerance (range 4.6% in Spain to 19.1% in Australia). Atopic females who had wheezed in the past 12 months, ever had asthma or were currently taking oral asthma medications were significantly more likely to report food allergy/intolerance. Participants from Scandinavia or Germany were significantly more likely than those from Spain to report food allergy/intolerance. Respondents who reported breathlessness as a food-related symptom were more likely to have wheezed in the past 12 months, to have asthma, use oral asthma medications, be atopic, have bronchial hyperreactivity, be older and reside in Scandinavia.Conclusion: Self-reported food allergy/intolerance differed significantly across multiple countries. The reasons for these differences were not explored in this study, but are likely to be largely due to cultural differences.Sponsorship: Rosalie Woods holds a postdoctoral research fellowship from the National Health and Medical Research Council of Australia (no. 9797/0883).European Journal of Clinical Nutrition (2001) 55, 298–304

Linkage of high-affinity IgE receptor gene with bronchial hyperreactivity, even in absence of atopy

Asthma is a manifestation of bronchial hyperreactivity (BHR) and forms part of the spectrum of atopic disease. Some pedigree studies of atopy have suggested linkage with the high-affinity IgE receptor (Fc epsilon RI beta) gene on chromosome 11q13, but others find no linkage. The molecular genetics of asthma and BHR have not been studied in the general population. We examined the genetic linkage of the Fc epsilon RI beta gene with clinical asthma and the underlying phenotypes of BHR (to methacholine) and atopy (defined by skinprick testing) in 123 affected sibling-pairs recruited from the general population. We found evidence of significant linkage of a highly polymorphic microsatellite marker in the fifth intron of the Fc epsilon RI beta gene to a diagnosis of asthma (18.0% excess of shared alleles, p = 0.002) and to BHR (21.7% excess of shared alleles, p = 0.001). Significant linkage was also observed in siblings sharing BHR when those with atopy were excluded (32.8% excess of shared alleles, p = 0.004). Atopy in the absence of BHR did not show significant linkage to the Fc epsilon RI beta gene (7.2% excess of shared alleles, p = 0.124). These findings suggest that mutations in the Fc epsilon RI beta gene or a closely linked gene influence the BHR underlying asthma, even in the absence of atopy.

Reported adverse food reactions overestimate true food allergy in the community

Objective: To determine the extent to which perceived adverse food reactions were associated with IgE mediated food allergy, as defined by skin prick testing (SPT).Design: A cohort epidemiological study. Participants underwent SPT to five common food allergens (cows milk, peanut mix, egg white, shrimp and whole grain wheat mix) and were asked whether they had ever suffered any food ‘illness/trouble’, and if so to list such food(s). A positive SPT was defined as wheal diameter of ≥3 mm. Cohens kappa (κ) was used to assess the agreement between SPT and self-reported reactions to food(s) which contained the allergen of interest.Setting: Randomly selected adults who took part in the follow-up of the European Community Respiratory Health Survey (ECRHS) in 1998.Subjects: The subjects were 457 adults aged 26–50 y.Results: Fifty-eight (13%) adults were sensitised to at least one food allergen whilst 99 adults (22%) reported illness to food(s) nearly always. However, only seven subjects who reported illness to a food also had a positive SPT to the same food. The prevalence of adverse food reactions associated with IgE mediated allergy in the adult general population would be less than 1.5% (7/457). The agreement between SPT and self-reported illness to food(s) was poor for cows milk (κ=0) and wheat (κ=0), slight for shrimp (κ=0.16) and egg white (κ=0.09) and fair for peanut mix (κ=0.37).Conclusions: There was little agreement between self-reported perceived illness to food(s) known to contain the food allergen of interest, and positive SPT, suggesting that most reactions are not due to IgE mediated food allergy.Sponsorship: The National Health and Medical Research Council of Australia (NH&MRC) funded this study. Rosalie Woods holds a postdoctoral research fellowship from the NH&MRC (#9797/0883).

Bronchus associated lymphoid tissue (BALT) in human lung: its distribution in smokers and non-smokers.

BACKGROUND--Bronchus associated lymphoid tissue (BALT) is a normal component of the lungs immune system in many animals and may be analogous to gut associated lymphoid tissue (GALT). This study aimed at assessing the nature and extent of BALT in human lung and determining whether its expression is induced within the human airway in response to smoking. METHODS--Paraffin embedded, formalin fixed full thickness bronchial wall sections were examined from 31 whole lung specimens derived from both smokers and non-smokers. Samples were taken from throughout the bronchial tree to include main stem bronchi, lobar bronchi and segmental bronchi, as well as first to third generation carinae. Standard 4 microns step sections were stained by haematoxylin and eosin and immunocytochemical methods to show foci of BALT. RESULTS--Examination of 256 airway sites detected 46 foci of BALT. These differed from those described in other mammals in being distributed throughout the bronchial tree, in being found in relation to bronchial glandular epithelium as well as luminal bronchial epithelium, and in lacking any accompanying M cells. Analysis by smoking status showed that the expression of BALT was significantly more common in smokers than non-smokers (82% (14/17) v 14% (2/14) respectively). CONCLUSIONS--The findings support the view that BALT in humans is an integral feature in a comparatively small proportion of lungs from non-smokers while being significantly more prominent in lungs from smokers. The tissue shows several important differences from that described in other mammals.

Meta-analysis of genome-wide association studies identifies ten loci influencing allergic sensitization

Allergen-specific immunoglobulin E (present in allergic sensitization) has a central role in the pathogenesis of allergic disease. We performed the first large-scale genome-wide association study (GWAS) of allergic sensitization in 5,789 affected individuals and 10,056 controls and followed up the top SNP at each of 26 loci in 6,114 affected individuals and 9,920 controls. We increased the number of susceptibility loci with genome-wide significant association with allergic sensitization from three to ten, including SNPs in or near TLR6, C11orf30, STAT6, SLC25A46, HLA-DQB1, IL1RL1, LPP, MYC, IL2 and HLA-B. All the top SNPs were associated with allergic symptoms in an independent study. Risk-associated variants at these ten loci were estimated to account for at least 25% of allergic sensitization and allergic rhinitis. Understanding the molecular mechanisms underlying these associations may provide new insights into the etiology of allergic disease.