[Show abstract][Hide abstract] ABSTRACT: Background:
Bronchial epithelial ciliary dysfunction is an important feature of asthma. We sought to determine the role in asthma of neutrophilic inflammation and NADPH oxidases in ciliary dysfunction.
Bronchial epithelial ciliary function was assessed by video-microscopy in fresh ex vivo epithelial strips from asthmatics stratified by their sputum cell differentials and in cultures from healthy controls and asthmatics. Bronchial epithelial oxidative damage was determined by 8-oxo-dG expression. NOX/DUOX expression was assessed in bronchial epithelial cells by microarrays, with NOX4, DUOX1/2 expression assessed in bronchial biopsies. Ciliary dysfunction following NADPH oxidase inhibition, using GKT137831, was evaluated in fresh epithelial strips from asthmatics and a murine model of ovalbumin sensitisation and challenge.
Ciliary beat frequency was impaired in asthmatics with sputum neutrophilia (n=11) versus those without (n=10) (5.8 [0.6] versus 8.8 [0.5]Hz; P=0.003) and was correlated with sputum neutrophil count (r=-0.70; P<0.001). Primary bronchial epithelial cells expressed DUOX1/2 and NOX4. 8-oxo-dG and NOX4, were elevated in neutrophilic versus non-neutrophilic asthmatics, DUOX1 was elevated in both, and DUOX2 was elevated in non-neutrophilic asthma in vivo. In primary epithelial cultures ciliary dysfunction did not persist, although NOX4 expression and reactive oxygen species generation was increased from subjects with neutrophilic asthma. GKT137831 both improved ciliary function in ex vivo epithelial strips (n=13), relative to the intensity of neutrophilic inflammation, and abolished ciliary dysfunction in the murine asthma model without a reduction in inflammation.
Ciliary dysfunction is increased in neutrophilic asthma associated with increased NOX4 expression and is attenuated by NADPH oxidase inhibition.
[Show abstract][Hide abstract] ABSTRACT: Background
Asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous diseases.
We sought to determine, in terms of their sputum cellular and mediator profiles, the extent to which they represent distinct or overlapping conditions supporting either the “British” or “Dutch” hypotheses of airway disease pathogenesis.
We compared the clinical and physiological characteristics and sputum mediators between 86 subjects with severe asthma and 75 with moderate-to-severe COPD. Biological subgroups were determined using factor and cluster analyses on 18 sputum cytokines. The subgroups were validated on independent severe asthma (n = 166) and COPD (n = 58) cohorts. Two techniques were used to assign the validation subjects to subgroups: linear discriminant analysis, or the best identified discriminator (single cytokine) in combination with subject disease status (asthma or COPD).
Discriminant analysis distinguished severe asthma from COPD completely using a combination of clinical and biological variables. Factor and cluster analyses of the sputum cytokine profiles revealed 3 biological clusters: cluster 1: asthma predominant, eosinophilic, high TH2 cytokines; cluster 2: asthma and COPD overlap, neutrophilic; cluster 3: COPD predominant, mixed eosinophilic and neutrophilic. Validation subjects were classified into 3 subgroups using discriminant analysis, or disease status with a binary assessment of sputum IL-1β expression. Sputum cellular and cytokine profiles of the validation subgroups were similar to the subgroups from the test study.
Sputum cytokine profiling can determine distinct and overlapping groups of subjects with asthma and COPD, supporting both the British and Dutch hypotheses. These findings may contribute to improved patient classification to enable stratified medicine.
Full-text · Article · Aug 2014 · The Journal of allergy and clinical immunology
[Show abstract][Hide abstract] ABSTRACT: Background
Asthma is characterized by variable airflow obstruction, airway inflammation, airway hyper-responsiveness and airway remodelling. Airway smooth muscle (ASM) hyperplasia is a feature of airway remodelling and contributes to bronchial wall thickening. We sought to investigate the expression levels of chemokines in primary cultures of ASM cells from asthmatics vs healthy controls and to assess whether differentially expressed chemokines (i) promote fibrocyte (FC) migration towards ASM and (ii) are increased in blood from subjects with asthma and in sputum samples from those asthmatics with bronchial wall thickening.
Chemokine concentrations released by primary ASM were measured by MesoScale Discovery platform. The chemokine most highly expressed by ASM from asthmatics compared with healthy controls was confirmed by ELISA, and expression of its cognate chemokine receptor by FCs was examined by immunofluorescence and flow cytometry. The role of this chemokine in FC migration towards ASM was investigated by chemotaxis assays.
Chemokine (C-C motif) ligand 2 (CCL2) levels were increased in primary ASM supernatants from asthmatics compared with healthy controls. CCR2 was expressed on FCs. Fibrocytes migrated towards recombinant CCL2 and ASM supernatants. These effects were inhibited by CCL2 neutralization. CCL2 levels were increased in blood from asthmatics compared with healthy controls, and sputum CCL2 was increased in asthmatics with bronchial wall thickening.
Airway smooth muscle-derived CCL2 mediates FC migration and potentially contributes to the development of ASM hyperplasia in asthma.
[Show abstract][Hide abstract] ABSTRACT: Background
The role of small airway obstruction in the clinical expression of asthma is incompletely understood. Objective
We tested the hypotheses that markers of small airway obstruction are associated with (i) increased asthma severity, (ii) impaired asthma control and quality of life and (iii) frequent exacerbations. Methods
Seventy-four adults with asthma and 18 healthy control subjects underwent impulse oscillometry (IOS), multiple breath inert gas washout (MBW), body plethysmography, single-breath determination of carbon monoxide uptake and spirometry. Patients completed the six-point Asthma Control Questionnaire (ACQ-6) and standardized Asthma Quality of Life Questionnaire [AQLQ(S)]. Asthma severity was classified according to the Global Initiative for Asthma (GINA) treatment steps. ResultsThe putative small airway obstruction markers S-acin, resistance at 5Hz minus resistance at 20Hz (R5-R20) and reactance area (AX) were not independently associated with asthma severity, control, quality of life or exacerbations. In contrast, markers of total (R5) and mean airway resistance of large and small airways (R20) were significantly higher in the severe asthma group compared with the mild-moderate group (0.47 vs. 0.37, P<0.05 for R5; 0.39 vs. 0.31, P<0.01 for R20). The strongest independent contributors to ACQ-6 score were R20 and forced expiratory volume in one second (% pred.), and the strongest independent contributors to AQLQ(S) score were R20 and forced vital capacity (% pred.). A history of one or more exacerbations within the previous year was independently associated with R20. Conclusions and Clinical RelevancePreviously reported markers of small airway obstruction do not appear to be independently associated with asthma disease expression. In contrast, the IOS parameter R20, a marker of mean airway resistance of both large and small airways, appears to have independent clinical significance. These observations require confirmation in prospective longitudinal studies.
No preview · Article · Dec 2013 · Clinical & Experimental Allergy
[Show abstract][Hide abstract] ABSTRACT: IgE sensitization to Aspergillus fumigatus and a positive sputum fungal culture result are common in patients with refractory asthma. It is not clear whether these patients would benefit from antifungal treatment.
We sought to determine whether a 3-month course of voriconazole improved asthma-related outcomes in patients with asthma who are IgE sensitized to A fumigatus.
Asthmatic patients who were IgE sensitized to A fumigatus with a history of at least 2 severe exacerbations in the previous 12 months were treated for 3 months with 200 mg of voriconazole twice daily, followed by observation for 9 months, in a double-blind, placebo-controlled, randomized design. Primary outcomes were improvement in quality of life at the end of the treatment period and a reduction in the number of severe exacerbations over the 12 months of the study.
Sixty-five patients were randomized. Fifty-nine patients started treatment (32 receiving voriconazole and 27 receiving placebo) and were included in an intention-to-treat analysis. Fifty-six patients took the full 3 months of medication. Between the voriconazole and placebo groups, there were no significant differences in the number of severe exacerbations (1.16 vs 1.41 per patient per year, respectively; mean difference, 0.25; 95% CI, 0.19-0.31), quality of life (change in Asthma Quality of Life Questionnaire score, 0.68 vs 0.88; mean difference between groups, 0.2; 95% CI, -0.05 to -0.11), or any of our secondary outcome measures.
We were unable to show a beneficial effect of 3 months of treatment with voriconazole in patients with moderate-to-severe asthma who were IgE sensitized to A fumigatus on either the rate of severe exacerbations, quality of life, or other markers of asthma control.
Preview · Article · Nov 2013 · The Journal of allergy and clinical immunology
[Show abstract][Hide abstract] ABSTRACT: Bacteria are often isolated in stable chronic obstructive pulmonary disease (COPD). Whether fungi are also commonly present and associated with clinical and pathological features of disease is uncertain. We investigated the frequency of filamentous fungal culture and IgE sensitization to Aspergillus fumigatus and the relationship to clinical outcomes in COPD subjects.COPD subjects were recruited to enter a 1 year observational study. Assessments of lung function, allergen testing and sputum analysis for inflammation, bacteria and fungus were undertaken in COPD subjects and healthy smoking and non-smoking controls.Filamentous fungi were cultured at baseline in 49% (63/128) of COPD subjects of which 75% (47/63) were A. fumigatus. Fungus was cultured in 3/22 controls (2 were A. fumigatus). The total sputum cell count and inhaled corticosteroid dosage were significantly increased in COPD patients with a positive filamentous fungal culture at baseline (p<0.05). Sensitization to A. fumigatus was present in 13% of COPD subjects and was associated with worse lung function (FEV1 % predicted 39% versus 51%; p=0.01), but not related to filamentous fungal culture.A. fumigatus sensitization is related to poor lung function. Positive filamentous fungal culture is a common feature of COPD. The clinical significance of this remains uncertain.
No preview · Article · Apr 2013 · European Respiratory Journal
[Show abstract][Hide abstract] ABSTRACT: Rationale: The relationship between airway inflammation and obesity in severe asthma is poorly understood. Objectives: We sought to determine the relationship between sputum mediator profiles, the distribution of eosinophilic inflammation and obesity in severe asthmatics. Methods: Clinical parameters and 8 mediators in sputum were assessed in 131 severe asthmatic subjects from a single centre categorised into lean, overweight and obese groups defined by their body mass index. In an independent group of severe asthmatics (n=45) and healthy controls (n=19) eosinophilic inflammation was enumerated in bronchial submucosa, blood and sputum and related to their body mass index. Measurements and Main Results: Sputum interleukin (IL)-5 geometric mean [95% confidence interval] (pg/ml) was elevated in the obese (1.5 [1.0-2.3]) compared to overweight (0.9 [0.7-1.1]; p=0.02) and lean (0.7 [0.5-1.0]; p=0.01) asthmatics and was correlated with the body mass index (r=0.29, p<0.001). There was no relationship between body mass index, the sputum cell count or other sputum mediators. In the bronchoscopy group the submucosal eosinophil number in the asthmatic subjects was correlated with body mass index (Spearman's rank correlation rs=0.38, p=0.013) and the median (interquartile range) number of submucosal eosinophils was increased in obese (19.4 [11.8-31.2]) cells/mm2 versus lean subjects (8.2 [5.4-14.6]) (p=0.006). There was no significant association between sputum or peripheral blood eosinophil counts and body mass index. Conclusion: Sputum IL-5 and submucosal eosinophils, but not sputum eosinophils are elevated in obese severe asthmatics. Whether specific anti-eosinophilic therapy is beneficial, or improved diet and lifestyle in obese asthma has anti-inflammatory effects beyond weight reduction, requires further study.
No preview · Article · Apr 2013 · American Journal of Respiratory and Critical Care Medicine
[Show abstract][Hide abstract] ABSTRACT: Background
Severe asthma is a heterogeneous disease and the relationship between airway inflammation and airway remodelling is poorly understood. We sought to define sputum mediator profiles in severe asthmatics categorised by CT-determined airway geometry and sputum differential cell counts.
In a single centre cross-sectional observational study we recruited 59 subjects with severe asthma that underwent sputum induction and thoracic CT. Quantitative CT analysis of the apical segment of the right upper lobe (RB1) was performed. Forty-one mediators in sputum samples were measured of which 21 mediators that were assessable in >50% of samples were included in the analyses.
Independent of airway geometry, sputum MMP9 and IL-1β were elevated in those groups with a high sputum neutrophil count while sputum ICAM was elevated in those subjects with a low sputum neutrophil count. In contrast, sputum CCL11, IL-1α and fibrinogen were different in groups stratified by both sputum neutrophil count and airway geometry. Sputum CCL11 concentration was elevated in subjects with a low sputum neutrophil count and high luminal and total RB1 area, whereas sputum IL1α was increased in subjects with a high sputum neutrophil count and low total RB1 area. Sputum fibrinogen was elevated in those subjects with RB1 luminal narrowing and in those subjects with neutrophilic inflammation without luminal narrowing.
We have demonstrated that sputum mediator profiling reveals a number of associations with airway geometry. Whether these findings reflect important biological phenotypes that might inform stratified medicine approaches requires further investigation.
Full-text · Article · Feb 2013 · Respiratory research
[Show abstract][Hide abstract] ABSTRACT: Introduction and objectives Asthma is an inflammatory disease that is treated with inhaled corticosteroids, but some patients manifest persistent symptoms despite this. Small airway dysfunction may account for treatment resistance in asthma. We hypothesised firstly that small airway disease is characterised by multiple and independent domains, and secondly that small airway biomarkers define a distinct phenotype of asthma with altered clinical disease expression.
Methods Ninety-six patients with asthma and eighteen healthy control subjects were recruited. Participants undertook spirometry, body plethysmography, single breath determination of carbon monoxide uptake in the lung, multiple breath inert gas washout and impulse oscillometry. Factor analysis was used to reduce multiple physiological variables to a smaller number of independent components. Hierarchical and k-means cluster analysis was used to classify asthma patients into groups based on physiological biomarkers.
Results Factor analysis showed that the measured physiological biomarkers could be reduced to three independent components, corresponding to abnormal lung mechanics (R5-R20 and reactance area), airflow obstruction (FEV1 [% pred.] and FVC [% pred.]) and ventilation heterogeneity (lung clearance index and Sacin). Cluster analysis classified the asthma patients into two groups. Patients in Cluster 1 exhibited multiple physiological abnormalities suggestive of small airway disease, including air trapping, ventilation heterogeneity and abnormal lung mechanics, as well as significant expiratory flow limitation. In contrast, patients in Cluster 2 had largely normal physiology. Patients in Cluster 1 exhibited increased clinical disease expression compared to patients in Cluster 2, with significantly worse median Asthma Control Questionnaire-6 (1.33 vs 1.17, p<0.05), Asthma Quality of Life (5.16 vs 5.97, p<0.01), visual analogue score (VAS) breathlessness (38.5 vs 19.5, p<0.05) and VAS wheeze (33.0 vs 12.0, p<0.05) scores.
Conclusion Small airway biomarkers define a distinct phenotype of asthma with multiple physiological abnormalities and increased disease expression. Future studies should examine the utility of screening for small airway disease at an early stage as a possible means of stratifying asthma therapy.
[Show abstract][Hide abstract] ABSTRACT: Asthma is increasing in prevalence worldwide. It is characterized by typical symptoms and variable airway obstruction punctuated with episodes of worsening symptoms known as exacerbations. Underlying this clinical expression of disease is airway inflammation and remodeling. Cytokines and their networks are implicated in the innate and adaptive immune responses driving airway inflammation in asthma and are modulated by host-environment interactions. Asthma is a complex heterogeneous disease, and the paradigm of Th2 cytokine-mediated eosinophilic inflammation as a consequence of allergic sensitization has been challenged and probably represents a subgroup of asthma. Indeed, as attention has switched to the importance of severe asthma, which represents the highest burden both to the patient and health care provider, there is an increasing recognition of inflammatory subphenotypes that are likely to be driven by different cytokine networks. Interestingly, these networks may be specific to aspects of clinical expression as well as inflammatory cell profiles and therefore present novel phenotype-specific therapeutic strategies. Here, we review the breadth of cytokines implicated in the pathogenesis of asthma and focus upon the outcomes of early clinical trials conducted using cytokines or cytokine-blocking therapies.
No preview · Article · Aug 2012 · Advances in clinical chemistry
[Show abstract][Hide abstract] ABSTRACT: Fungal sensitization is common in severe asthma, but the clinical relevance of this and the relationship with airway colonization by fungi remain unclear. The range of fungi that may colonize the airways in asthma is unknown.
To provide a comprehensive analysis on the range of filamentous fungi isolated in sputum from people with asthma and report the relationship with their clinico-immunological features of their disease.
We recruited 126 subjects with a diagnosis of asthma, 94% with moderate-severe disease, and 18 healthy volunteers. At a single stable visit, subjects underwent spirometry; sputum fungal culture and a sputum cell differential count; skin prick testing to both common aeroallergens and an extended fungal panel; specific IgE to Aspergillus fumigatus. Fungi were identified by morphology and species identity was confirmed by sequencing. Four patients had allergic bronchopulmonary aspergillosis.
Forty-eight percent of asthma subjects were IgE-sensitized to one fungal allergen and 22% to ≥ 2. Twenty-seven different taxa of filamentous fungi were isolated from 54% of their sputa, more than one species being detected in 17%. This compared with 3 (17%) healthy controls culturing any fungus (P < 0.01). Aspergillus species were most frequently cultured in isolation followed by Penicillium species. Post-bronchodilator FEV (1) (% predicted) in the subjects with asthma was 71(± 25) in those with a positive fungal culture vs. 83 (± 25) in those culture-negative, (P < 0.01).
Numerous thermotolerant fungi other than A. fumigatus can be cultured from sputum of people with moderate-to-severe asthma; a positive culture is associated with an impaired post-bronchodilator FEV (1) , which might be partly responsible for the development of fixed airflow obstruction in asthma. Sensitization to these fungi is also common.
[Show abstract][Hide abstract] ABSTRACT: Variability of peak flow measurements has been related to clinical outcomes in asthma. We hypothesized that the entropy, or information content, of airway impedance over short time scales may predict asthma exacerbation frequency.Sixty-six patients with severe asthma and thirty healthy control subjects underwent impulse oscillometry at baseline, following a deep exhalation manoeuvre, and following bronchodilator administration. On each occasion, airway impedance parameters were measured at 0.2 second intervals for 150 seconds, yielding a time series, which was then subjected to Sample Entropy analysis.Airway impedance, and Sample Entropy of impedance, was increased in asthmatic patients compared to healthy controls. In a logistic regression model, Sample Entropy of R5-R20, a marker of the fluctuation of the heterogeneity of airway constriction over time, was the variable most strongly associated with the frequent exacerbation phenotype (odds ratio of 3.23 for every 0.1 increase in Sample Entropy).Increased airway impedance and Sample Entropy of impedance is associated with the frequent exacerbation phenotype. Prospective studies are required to assess their predictive value.
Full-text · Article · Mar 2012 · European Respiratory Journal