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Background Asthma is a chronic inflammatory airway disease, associated with episodes of exacerbations. Therapy with inhaled corticosteroids ( ICS) targets airway inflammation, which aims to maintain and restore asthma control. Clinical features are only modestly associated with airways inflammation. Therefore, we hypothesized that exhaled volatile metabolites identify longitudinal changes between clinically stable episodes and loss of asthma control. Objectives To determine whether exhaled volatile organic compounds ( VOCs) as measured by gas-chromatography/mass-spectrometry ( GC/ MS) and electronic nose ( eNose) technology discriminate between clinically stable and unstable episodes of asthma. Methods Twenty-three patients with (partly) controlled mild to moderate persistent asthma using ICS were included in this prospective steroid withdrawal study. Exhaled metabolites were measured at baseline, during loss of control and after recovery. Standardized sampling of exhaled air was performed, after which samples were analysed by GC/ MS and eNose. Univariate analysis of covariance ( ANCOVA), followed by multivariate principal component analysis ( PCA) was used to reduce data dimensionality. Next paired t tests were utilized to analyse within-subject breath profile differences at the different time-points. Finally, associations between exhaled metabolites and sputum inflammation markers were examined. Results Breath profiles by eNose showed 95% (21/22) correct classification for baseline vs loss of control and 86% (19/22) for loss of control vs recovery. Breath profiles using GC/ MS showed accuracies of 68% (14/22) and 77% (17/22) for baseline vs loss of control and loss of control vs recovery, respectively. Significant associations between exhaled metabolites captured by GC/ MS and sputum eosinophils were found (Pearson r≥.46, P<.01). Conclusions & Clinical Relevance Loss of asthma control can be discriminated from clinically stable episodes by longitudinal monitoring of exhaled metabolites measured by GC/ MS and particularly eNose. Part of the uncovered biomarkers was associated with sputum eosinophils. These findings provide proof of principle for monitoring and identification of loss of asthma control by breathomics. [ABSTRACT FROM AUTHOR]

Background Assessing future risk of exacerbations is an important component of asthma management. Existing studies have investigated short- but not long-term risk. Problematic asthma patients with unfavorable long-term disease trajectory and persistently frequent severe exacerbations need to be identified early to guide treatment. Aim To identify distinct trajectories of severe exacerbation rates among 'problematic asthma' patients and develop a risk score to predict the most unfavorable trajectory. Methods Severe exacerbation rates over five years for 177 'problematic asthma' patients presenting to a specialist asthma clinic were tracked. Distinct trajectories of severe exacerbation rates were identified using group-based trajectory modeling. Baseline predictors of trajectory were identified and used to develop a clinical risk score for predicting the most unfavorable trajectory. Results Three distinct trajectories were found: 58.5% had rare intermittent severe exacerbations ('infrequent'), 32.0% had frequent severe exacerbations at baseline but improved subsequently ('nonpersistently frequent'), and 9.5% exhibited persistently frequent severe exacerbations, with the highest incidence of near-fatal asthma ('persistently frequent'). A clinical risk score composed of ≥2 severe exacerbations in the past year (+2 points), history of near-fatal asthma (+1 point), body mass index ≥25kg/m2 (+1 point), obstructive sleep apnea (+1 point), gastroesophageal reflux (+1 point), and depression (+1 point) was predictive of the 'persistently frequent' trajectory (area under the receiver operating characteristic curve: 0.84, sensitivity 72.2%, specificity 81.1% using cutoff ≥3 points). The trajectories and clinical risk score had excellent performance in an independent validation cohort. Conclusions Patients with problematic asthma follow distinct illness trajectories over a period of five years. We have derived and validated a clinical risk score that accurately identifies patients who will have persistently frequent severe exacerbations in the future. [ABSTRACT FROM AUTHOR]

Background Acute worsening of asthma symptoms (exacerbation) is predominantly triggered by respiratory viruses, with influenza causing the most severe exacerbations. The lack of an adequate animal model hampers mechanistic insight and the development of new therapeutics. Aim We developed and characterized a robust, consistent, and reproducible mouse model of severe exacerbation of chronic allergic asthma. Methods Chronic allergic airway inflammation was induced following a house dust mite ( HDM) sensitization protocol. HDM-sensitized mice and controls were infected with influenza virus A/X31 H3N2 and either or not treated with inhaled fluticasone propionate ( FP), systemic corticosteroids (Pred), or anti- IL-5. Mice were killed at different time points after infection: Cellular accumulation and cytokines levels in the airways, PenH as a measure of airway hyper-responsiveness ( AHR), and lung histology and viral replication were assessed. Results Infection with low-dose A/X31 H3N2 led to prolonged deterioration of lung function, aggravated mucus production, peri-vascular, peri-bronchial, and allergic inflammation that was unresponsive to inhaled corticosteroids, but responsive to systemic corticosteroids. The exacerbation was preceded at 14 h after virus exposure by a marked innate, but no Th2 and Th1 response subsequently followed by enhanced numbers of eosinophils, neutrophils, dendritic, and T cells into the lung lumen, parenchyma, and draining lymph nodes in HDM-sensitized mice. Anti- IL-5 treatment attenuated eosinophils and prevented the X31-induced exacerbation. Conclusions Together, these findings indicate that an early innate response that involves eosinophils underlies the exacerbation. This model recapitulates all major features of severe asthma exacerbations and can serve to discern driving mechanisms and promote the development of novel therapeutics. [ABSTRACT FROM AUTHOR]

Background Bradykinin ( BK) mediates acute allergic asthma and airway remodelling. Nuclear factor-kappa B ( NF- kB) is potentially involved in BK B2 receptor (B2R) regulation. Objective In this observational cross-sectional study, B2R and NF- kB expression was evaluated in bronchial biopsies from mild asthmatics (after diluent/allergen challenge) and healthy controls, examining the role of NF- kB in B2R expression in primary human fibroblasts from normal and asthmatic subjects ( HNBFb and HABFb). Methods B2R and NF- kB (total and nuclear) expression was analysed by immunohistochemistry in biopsies from 10 mild intermittent asthmatics (48 h after diluent/allergen challenge) and 10 controls undergoing bronchoscopy. B2R co-localization in 5B5+ and α SMA+ mesenchymal cells was studied by immunofluorescence/confocal microscopy, and B2R expression in HABFb/ HNBFb incubated with interleukin ( IL)-4/ IL-13 with/without BK, and after NF- kB inhibitor, by Western blotting. Results Bronchial mucosa B2R and nuclear NF- kB expression was higher in asthmatics after diluent (B2R only) and allergen challenge than in controls ( P < 0.05), while B2R and NF-kB (total and nuclear) increased after allergen compared with after diluent ( P < 0.05). Allergen exposure increased B2R expression in 5B5+ and α SMA+ cells. Constitutive B2R protein expression was higher in HABFb than in HNBFb ( P < 0.05) and increased in both cell types after IL-13 or IL-4/ IL-13 and BK treatment. This increase was suppressed by a NF- kB inhibitor ( P < 0.05). Conclusions & Clinical Relevance Bronchial B2R expression is constitutively elevated in allergic asthma and is further increased after allergen exposure together with NF- kB expression. NF- kB inhibitor blocked IL-4/ IL-13-induced increase in B2R expression in cultured fibroblasts, suggesting a role as potential anti-asthma drug. [ABSTRACT FROM AUTHOR]

Background Epidemiologic studies have shown that patients with severe asthma have increased risk of pulmonary embolism, in particular patients with frequent asthma exacerbations. Therefore, we hypothesized that asthma exacerbations are associated with increased haemostatic activity. Objective To investigate whether induced loss of asthma control is associated with changes in coagulation and fibrinolytic parameters in peripheral blood. Methods We performed a prospective, inhaled steroid withdrawal study in 23 patients with moderate to moderately severe asthma, consisting of a baseline visit and a visit after loss of asthma control. During the visits, we measured asthma control questionnaire ( ACQ), atopy, lung function, inflammatory markers (eosinophils and neutrophils), and haemostatic parameters in plasma. Results Complete cessation of inhaled corticosteroids led to a loss of asthma control in 22 of 23 patients. We found increased asthma symptoms ( ACQ 0.9 vs. 2.9, P < 0.01), significantly reduced lung function (forced expiratory volume in 1 s ( FEV1) 3.51L vs. 3.13L, P < 0.01) and increased levels of eosinophils in plasma (0.26 × 10E9/L vs. 0.16 × 10E9/L, P = 0.03) in patients after loss of asthma control. However, we observed no significant changes in the coagulation and fibrinolysis parameters. Conclusion Loss of asthma control after cessation of inhaled corticosteroids does not lead to increased haemostatic activation in patients with moderate to moderately severe asthma. This suggests that more severe inflammation or additional risk factors are required for activation of coagulation or reduction of fibrinolysis in asthma. [ABSTRACT FROM AUTHOR]

Background House dust contains mite allergens as well as bacterial products such as lipopolysaccharide ( LPS). Asthma exacerbations are associated with the level of exposure to allergens and LPS. LPS can potentiate allergen effects in steroid-naïve patients. Long-acting β2-agonists ( LABA) were shown to inhibit LPS-induced bronchial inflammation in healthy volunteers. The aim of this study was to assess the effect of LPS on the allergen-induced eosinophilic inflammation [primary endpoints: eosinophil counts and eosinophil cationic protein ( ECP)] induced by bronchial instillation of house dust mite ( HDM) in patients with asthma on maintenance treatment with inhaled corticosteroids ( ICS). Methods Thirty-two nonsmoking asthmatics with HDM allergy were treated with run-in medication (fluticasone propionate 100 μg bid) during 2 weeks before the study day. All patients underwent bronchial challenge with HDM, and half of them were randomized to receive additional LPS. Both groups were randomized to receive pretreatment with a single inhalation of 100 μg salmeterol 30 min before bronchial segmental challenge. Six hours later, bronchoalveolar lavage ( BAL) was collected for leukocyte cell count, differentials, and cellular activation markers. Results Challenge with HDM/ LPS induced a significant increase in eosinophil cationic protein ( P = 0.036) and a trend toward an increase in BALF eosinophils as compared to HDM challenge. Conclusion Lipopolysaccharide promotes eosinophilic airway inflammation in patients with asthma despite being on maintenance treatment with ICS. [ABSTRACT FROM AUTHOR]

Background Asthma and atopy share common characteristics including type 2 helper-T-cell-mediated inflammation. However, only asthma is associated with variable airways obstruction. The complex cellular and molecular pathways distinguishing asthma and atopy can now be captured by transcriptomic analysis ( RNA-Seq). We hypothesized that the transcriptomic profile of airway smooth muscle ( ASM) distinguishes atopic asthma from atopic healthy controls. First, we compared the ASM transcriptomic profiles of endobronchial biopsies between glucocorticoid-free, atopic asthma patients, and atopic and nonatopic healthy controls. Second, we investigated the association between ASM transcriptomic profiles and airway function. Methods Twelve asthma patients and 12 control subjects (six atopic, six nonatopic) underwent bronchoscopy. RNA of laser-dissected ASM from 96 bronchial biopsy specimens was sequenced with Roche GS FLX. Gene networks were identified using Ingenuity Pathway Analysis. RNA-Seq reads were assumed to follow a negative binomial distribution. With the current sample size, the estimated false discovery rate was approximately 1%. Results One hundred and seventy four ASM genes were differentially expressed between asthma patients and atopic controls, 108 between asthma patients and nonatopic controls, and 135 between atopic and nonatopic controls. A set of eight genes discriminated asthma patients from nonasthmatic controls, irrespective of atopy. Four of these genes ( RPTOR, VANGL1, FAM129A, LEPREL1) were associated with airway hyper-responsiveness ( P < 0.05). Conclusion Airway smooth muscle from asthma patients can be distinguished from that of atopic and nonatopic control subjects by a specific gene expression profile, which is associated with airway hyper-responsiveness. [ABSTRACT FROM AUTHOR]

Background Although asthma is characterized by variable airways obstruction, most studies of asthma phenotypes are cross-sectional. The stability of phenotypes defined either by biomarkers or by physiological variables was assessed by repeated measures over 1 year in the Pan-European BIOAIR cohort of adult asthmatics. Methods A total of 169 patients, 93 with severe asthma ( SA) and 76 with mild-to-moderate asthma ( MA), were examined at six or more visits during 1 year. Asthma phenotype clusters were defined by physiological variables (lung function, reversibility and age of onset of the disease) or by biomarkers (eosinophils and neutrophils in induced sputum). Results After 1 year of follow-up, the allocation to clusters was changed in 23.6% of all asthma patients when defined by physiological phenotypes and, remarkably, in 42.3% of the patients when stratified according to sputum cellularity ( P = 0.034). In the SA cohort, 30% and 48.6% of the patients changed allocation according to physiological and biomarker clustering, respectively. Variability of phenotypes was not influenced by change in oral or inhaled corticosteroid dose, nor by the number of exacerbations. Lower stability of single and repeated measure was found for all evaluated biomarkers (eosinophils, neutrophils and Fe NO) in contrast to good stability of physiological variables ( FEV1), quality of life and asthma control. Conclusion Phenotypes determined by biomarkers are less stable than those defined by physiological variables, especially in severe asthmatics. The data also imply that definition of asthma phenotypes is improved by repeated measures to account for fluctuations in lung function, biomarkers and asthma control. [ABSTRACT FROM AUTHOR]

Introduction To prevent extensive pulmonary lesions in submerged oxygen divers lung function like the forced vital capacity (FVC) or the diffusing capacity for carbon monoxide (D L, co) are used to monitor pulmonary oxygen toxicity (POT). As the diffusing capacity for nitric oxide (D L, no) measures more accurately the membrane diffusing capacity compared to D L, co we hypothesized that DL, no is superior in monitoring the onset of physiological changes indicative of POT as compared to DL, co or FVC. Method 26 healthy divers (mean age 30.7 ± 6.2 years) made two submerged dives to 190 kPa for 1 h on two randomized separate days, whilst breathing 100% oxygen or compressed air. FVC, DL, no, DL, co and alveolar volume (VA) were measured 6 times during a 26-h period. Results Up to 8 h no significant differences in outcomes were found between the oxygen and air dives. However, at 8 h after the oxygen dives there was a significant reduction in DL, no, DL, co and VA as compared with air dives. In contrast, the reduction in FVC was significantly greater after the air dive. At 22 h there were no longer differences in outcomes between the dives. Conclusions These data show that DL, no and DL, co are significantly reduced 8 h after submerged oxygen dives as compared to similar air dives. Together with the reduction in VA this may be indicative of interstitial edema as an early sign of POT. Our data warrant validation of the superiority of DL, no and DL, co over FVC in the practical monitoring of divers. [ABSTRACT FROM AUTHOR]

Background Exacerbations represent a major source of morbidity and mortality in asthma and are a prominent feature of poorly controlled, difficult-to-treat disease. Objective The goal of our study was to provide a detailed characterization of the frequent exacerbator phenotype and to identify risk factors associated with frequent and seasonal exacerbations. Methods Ninety-three severe asthmatics ( SA) and 76 mild-to-moderate patients ( MA) were screened and prospectively followed up for 1 year ( NCT00555607). Medical history, baseline clinical data and biomarkers were used to assess risk factors for frequent exacerbations. Results During the study, 104 exacerbations were recorded in the SA group and 18 in the MA. Frequent exacerbators were characterized by use of higher doses of inhaled (1700 vs. 800 μg) and oral (6.7 vs. 1.7 mg) glucocorticosteroids, worse asthma control ( ACQ score 2.3 vs. 1.4), lower quality of life ( SGRQ score 48.5 vs. 33.3), higher sputum eosinophils (25.7% vs. 8.2%) and a more rapid decline in FEV1/ FVC ratio (−0.07 vs. −0.01 Δ FEV1/ FVC, frequent vs. non-frequent, respectively, P < 0.05). Exhaled NO > 45 p.p.b. and a history of smoking were associated with an increased risk of frequent exacerbations (odds ratios: 4.32 and 2.90 respectively). Conclusion and Clinical Relevance We were able to distinguish and characterize a subphenotype of asthma subjects - frequent exacerbators - who are significantly more prone to exacerbations. Patients with Fe NO > 45 p.p.b. and a history of smoking are at increased risk of frequent exacerbations and require careful monitoring in clinical practice. [ABSTRACT FROM AUTHOR]

Exhaled air contains many volatile organic compounds (VOCs) that are the result of normal and disease-associated metabolic processes anywhere in the body. Different omics techniques can assess the pattern of these VOCs. One such omics technique suitable for breath analysis is represented by electronic noses ( eNoses), providing fingerprints of the exhaled VOCs, called breathprints. Breathprints have been shown to be altered in different disease states, including in asthma and COPD. This review describes the current status on clinical validation and application of breath analysis by electronic noses in the diagnosis and monitoring of chronic airways diseases. Furthermore, important methodological issues including breath sampling, modulating factors and incompatibility between eNoses are raised and discussed. Next steps towards clinical application of electronic noses are provided, including further validation in suspected disease, assessment of the influence of different comorbidities, the value in longitudinal monitoring of patients with asthma and COPD and the possibility to predict treatment responses. Eventually, a Breath Cloud may be constructed, a large database containing disease-specific breathprints. When collaborative efforts are put into optimization of this technique, it can provide a rapid and non-invasive first line diagnostic test. [ABSTRACT FROM AUTHOR]

Rationale Adult-onset asthma differs from childhood-onset asthma in many respects. It is more heterogeneous, often severe and frequently associated with loss of lung function. To identify underlying mechanisms of adult-onset asthma and to capture predictors of disease progression, detailed characterization and phenotyping is necessary. Objectives To characterize adult-onset asthma and identify subphenotypes of adult-onset asthma. Methods A cohort of 200 patients with adult-onset (>18 year) asthma (age 54 (26-75) year) was recruited from one academic and three nonacademic pulmonary outpatient clinics in Amsterdam, the Netherlands. These patients were fully characterized with respect to clinical, functional and inflammatory markers. After data reduction, K-means nonhierarchical cluster analysis was performed to identify clusters of adult-onset asthma. Measurements and main results Patients with adult-onset asthma were predominately female (61%) and nonatopic (55%). Within this group of patients were identified three clusters of adult-onset asthma. Cluster 1 ( n = 69) consisted of patients with severe eosinophilic inflammation-predominant asthma and persistent airflow limitation despite high-intensity anti-inflammatory treatment, with relatively low symptom scores. The second cluster was characterized by obese women with frequent symptoms, high healthcare utilization and low sputum eosinophils. The third cluster consisted of patients with mild-to-moderate, well-controlled asthma with normal lung function and low inflammatory markers. Repeatability accuracy was 98.2%. Conclusions Amongst patients with adult-onset asthma, three subphenotypes can be identified with distinct clinical and inflammatory characteristics. These subphenotypes help to understand the underlying pathobiology and provide clinicians with directions for personalized management. [ABSTRACT FROM AUTHOR]

Background Airway inflammation in asthma involves innate immune responses. Toll-like receptors ( TLRs) and thymic stromal lymphopoietin ( TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics' both large and small airways has not been investigated. Objective To analyse the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and non-smoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection. Methods Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 non-smokers, 11 smokers) and nine deceased control subjects ( DCtrl). TLRs were also measured in 18 mild asthmatics ( MA) and 12 healthy controls ( HCtrl). M. pneumoniae and C. pneumoniae in autopsy lung tissue were analysed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects. Results Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than non-smoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression. Conclusions and Clinical Relevance Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths. [ABSTRACT FROM AUTHOR]

Background Patients with asthma have on average a more rapid decline in FEV1 as compared with the general population. Recent cluster analysis has revealed different asthma phenotypes that can be distinguished by age of onset and reversibility of airflow limitation. Objective This study aimed at detecting risk factors associated with persistent airflow limitation in patients with the adult onset asthma phenotype. Methods We recruited 88 patients with adult onset (≥ 18 years) asthma from an academic pulmonary outpatient clinic in the Netherlands. The associations of age, age of asthma onset, asthma duration, gender, race, atopy, smoking packyears, BMI, use of oral corticosteroids with post-bronchodilator FEV1/ FVC were investigated. Results Multiple linear regression analysis showed an association of absence of atopy ( r = −0.27, B = −0.26, P = 0.01) and male gender ( r = 0.31, B = 0.30, P = 0.004) with post-bronchodilator FEV1/ FVC. Multiple logistic regression analysis showed that male patients were 10.8 ( CI: 2.6-45.2) times the odds than women to have an FEV1/ FVC < 0.7, and non-atopic patients were 5.2 ( CI: 1.3-20.3) times the odds to have an FEV1/ FVC < 0.7 than atopic patients. Conclusions and Clinical Relevance We conclude that in patients with adult onset asthma, male gender and absence of atopy are associated with persistent airflow limitation. This might suggest that amongst patients with adult onset asthma, non-atopic male patients are at increased risk of accelerated decline in lung function. [ABSTRACT FROM AUTHOR]

Background Altered deposition of extracellular matrix ( ECM) in the airway smooth muscle ( ASM) layer as observed in asthma may influence ASM mechanical properties. We hypothesized that ECM in ASM is associated with airway function in asthma. First, we investigated the difference in ECM expression in ASM between asthma and controls. Second, we examined whether ECM expression is associated with bronchoconstriction and bronchodilation in vivo. Methods Our cross-sectional study comprised 19 atopic mild asthma patients, 15 atopic and 12 nonatopic healthy subjects. Spirometry, methacholine responsiveness, deep-breath-induced bronchodilation (Δ Rrs) and bronchoscopy with endobronchial biopsies were performed. Positive staining of elastin, collagen I, III and IV, decorin, versican, fibronectin, laminin and tenascin in ASM was quantified as fractional area and mean density. Data were analysed using Pearson's or Spearman's correlation coefficient. Results Extracellular matrix expression in ASM was not different between asthma and controls. In asthmatics, fractional area and mean density of collagen I and III were correlated with methacholine dose-response slope and Δ Rrs, respectively ( r = 0.71, P < 0.01; r = 0.60, P = 0.02). Furthermore, ASM collagen III and laminin in asthma were correlated with FEV1 reversibility ( r = −0.65, P = 0.01; r = −0.54, P = 0.04). Conclusion In asthma, ECM in ASM is related to the dynamics of airway function in the absence of differences in ECM expression between asthma and controls. This indicates that the ASM layer in its full composition is a major structural component in determining variable airways obstruction in asthma. [ABSTRACT FROM AUTHOR]

Summary Background Fixed airflow limitation can be found both in asthma and chronic obstructive pulmonary disease (COPD), posing a day-to-day diagnostic challenge. Objective We aimed to determine the external validity of metabolomic analysis of exhaled air by electronic nose for distinguishing asthma and COPD in patients with fixed airways obstruction. Methods One hundred patients were included in a cross-sectional design: 60 asthma patients: 21 with fixed airways obstruction (fixed asthma), 39 with reversible airways obstruction (classic asthma) and 40 COPD patients (GOLD stages II-III). Standardized sampling of exhaled breath was performed and volatile organic compounds were captured using an electronic nose resulting in breathprints. External validity in newly recruited patients (validation sets) was tested using a previous and independent training set. Breathprints were analysed by principal component and canonical discriminant analysis and area under the curve (AUC) of receiver operating characteristic curves. Results External validity of breathprints showed 88% accuracy for distinguishing fixed asthma from COPD (AUC 0.95, 95% CI 0.84-1.00, sensitivity 85%, specificity 90%) and 83% for classic asthma (AUC 0.93, 95% CI 0.87-1.00, sensitivity 91%, specificity 90%) (both P<0.001). Discriminative accuracy was not confounded by current smoking. Conclusions and Clinical Relevance External validation of exhaled breath molecular profiling shows high accuracy in distinguishing asthma and COPD in newly recruited patients with fixed airways obstruction. Exhaled air analysis may therefore reduce misdiagnosis in obstructive airways diseases, potentially leading to more appropriate management. Cite this as: N. Fens, A. C. Roldaan, M. P. van der Schee, R. J. Boksem, A. H. Zwinderman, E. H. Bel and P. J. Sterk, Clinical & Experimental Allergy, 2011 (41) 1371-1378. [ABSTRACT FROM AUTHOR]

Summary [ABSTRACT FROM AUTHOR]

Background: Respiratory viruses may persist in the airways of asthmatics between episodes of clinical worsening. We hypothesized that patients with clinically stable, severe asthma exhibit increased and more prolonged viral presence in the airways as compared to mild asthmatics and healthy controls. Methods: Thirty-five subjects (no cold symptoms >4 weeks) entered a 12-week prospective study using three groups: clinically stable mild asthma (GINA 2) (n = 12, age 34.1 ± 13.4 year), severe asthma (GINA 4) (n = 12, age 49.3 ± 14.8 year) and healthy controls (n = 11, age 37.9 ± 14.2 year). All subjects underwent spirometry and completed a written questionnaire on asthma symptoms at baseline. Nasal and throat swabs, induced sputum samples, exhaled breath condensate and gelatine-filtered expired air were analysed at 0, 6 and 12 weeks by a multiplex real-time PCR assay for 14 respiratory viruses using adequate positive and negative controls. Results: Thirty-two of 525 patient assessments (6%) showed a virus-positive sample. Among the 14 respiratory viruses examined, HRV, adenovirus, respiratory syncytial virus, parainfluenza 3&4, human bocavirus, influenza B and coronavirus were detected. When combining all sampling methods, on average 18% of controls and 30% of mild and severe asthmatics were virus positive, which was not different between the groups (P = 0.34). The longitudinal data showed a changing rather than persistent viral presence over time. Conclusion: Patients with clinically stable asthma and healthy controls have similar detection rates of respiratory viruses in samples from nasopharynx, sputum and exhaled air. This indicates that viral presence in the airways of stable (severe) asthmatics varies over time rather than being persistent. [ABSTRACT FROM AUTHOR]

Background IgE and its high-affinity receptor FcℇRI play an important role in allergy and asthma. The distribution of FcℇRI expression in the airways and within the airway wall, however, is largely unknown. Objective In this study, we aimed to map the distribution of FcℇRI in different layers of large airways (LA) and small airways (SA) in lung tissue from non-smoking and smoking patients who died of asthma [fatal asthma (FA)] and non-smoking controls (CTR). Methods Postmortem lung tissue from 24 cases of non-smoking FA, 13 smoking FA patients and from 19 subjects who died of non-pulmonary causes (CTR) was immunohistochemically stained for FcℇRI and AA1 (mast cell tryptase marker). The expression of these markers was analysed in inner, muscle, and outer layers of both LA and SA by image analysis. Results FcℇRI expression was higher in non-smoking and smoking FA compared with CTR in the inner and outer layer of SA. In the outer layer of LA, FcℇRI expression was higher in non-smoking FA compared with CTR. AA1 was higher in non-smoking FA compared with smoking FA and CTR in the outer layer of the SA, which was correlated with FcℇRI in this layer. Conclusion Our results show that the expression of FcℇRI is higher in both LA and SA in FA compared with CTR. These differences are predominantly found in the outer layer where they can be attributed in part to the increased mast cell numbers. These results indicate an increased capacity to mount IgE-mediated reactions in FA, both in LA and SA. Cite this as: I. den Otter, L. F. F. Silva, A. L. N. Carvalho, R. C. Pires-Neto, R. Annoni, D. S. Ferreira, I. Bajema, A. van Schadewijk, K. F. Rabe, M. Dolhnikoff, P. J. Sterk, P. S. Hiemstra and T. Mauad, Clinical & Experimental Allergy, 2010 (40) 1473–1481. [ABSTRACT FROM AUTHOR]

Background: Anti-IgE, omalizumab, inhibits the allergen response in patients with asthma. This has not been directly related to changes in inflammatory conditions. We hypothesized that anti-IgE exerts its effects by reducing airway inflammation. To that end, the effect of anti-IgE on allergen-induced inflammation in bronchial biopsies in 25 patients with asthma was investigated in a randomized, double-blind, placebo-controlled study. Methods: Allergen challenge followed by a bronchoscopy at 24 h was performed at baseline and after 12 weeks of treatment with anti-IgE or placebo. Provocative concentration that causes a 20% fall in forced expiratory volume in 1 s (PC20) methacholine and induced sputum was performed at baseline, 8 and 12 weeks of treatment. Changes in the early and late responses to allergen, PC20, inflammatory cells in biopsies and sputum were assessed. Results: Both the early and late asthmatic responses were suppressed to 15.3% and 4.7% following anti-IgE treatment as compared with placebo ( P < 0.002). This was paralleled by a decrease in eosinophil counts in sputum (4–0.5%) and postallergen biopsies (15–2 cells/0.1 mm2) ( P < 0.03). Furthermore, biopsy IgE+ cells were significantly reduced between both the groups, whereas high-affinity IgE receptor and CD4+ cells were decreased within the anti-IgE group. There were no significant differences for PC20 methacholine. Conclusion: The response to inhaled allergen in asthma is diminished by anti-IgE, which in bronchial mucosa is paralleled by a reduction in eosinophils and a decline in IgE-bearing cells postallergen without changing PC20 methacholine. This suggests that the benefits of anti-IgE in asthma may be explained by a decrease in eosinophilic inflammation and IgE-bearing cells. [ABSTRACT FROM AUTHOR]

Background: Asthma and obesity are associated disorders, but the contribution of obesity to difficult-to-treat asthma as well as the mechanisms responsible for this relationship are unclear. The aim of this study was to investigate the relationship between obesity (body mass index ≥ 30) and factors related with asthma severity in patients with difficult-to-treat asthma. Methods: One hundred and thirty-six nonsmoking asthmatic adults with persistent symptoms despite high doses of inhaled or oral corticosteroids and long-acting bronchodilators were studied [70% female, median (range) age 44.6 (18–75) years, 32% on daily oral corticosteroids]. The association between obesity, lung function parameters [forced expiratory volume in 1 s (FEV1), functional residual capacity/total lung capacity (FRC/TLC)], inflammatory markers [blood eosinophils, sputum eosinophils and neutrophils, exhaled nitric oxide (FENO), airway hyperresponsiveness, C-reactive protein (CRP)] and aggravating co-morbid factors (severe chronic sinus disease, gastro-esophageal reflux, recurrent respiratory infections, psychopathology and obstructive sleep apnea) was investigated. Results: Obese patients ( n = 29) had a higher FEV1%pred ( P = 0.05) and a lower FRC/TLC%pred ( P < 0.01) compared with nonobese patients ( n = 107). Body mass index was inversely related with sputum eosinophils ( r = −0.36, P < 0.01) and FENO ( r = −0.30, P < 0.01). Obese patients had an increased risk for gastro-esophageal reflux (OR = 2.3) and sleep apnea (OR = 3.1). Conclusion: Obesity in patients with difficult-to-treat asthma is inversely related with sputum eosinophils and FENO, and positively associated with the presence of co-morbid factors and reduced lung volumes. This suggests that other factors than airway inflammation alone explain the relationship between obesity and asthma severity. [ABSTRACT FROM AUTHOR]

Background House dust mite (HDM) is the most common aeroallergen causing sensitization in many Western countries and is often used in allergen inhalation challenges. The concentration of inhaled allergen causing an early asthmatic reaction [provocative concentration of inhaled allergen causing a 20% fall of forced expiratory volume in 1 s (FEV1)(PC20 allergen)] needs to be predicted for safety reasons to estimate accurately the severity of allergen-induced airway responsiveness. This can be accomplished by using the degree of non-specific airway responsiveness and skin sensitivity to allergen. Objective We derived prediction equations for HDM challenges using PC20 histamine or PC20 methacholine and skin sensitivity data obtained from patients with mild to moderate persistent asthma and validated these equations in an independent asthma population. Methods PC20 histamine or PC20 methacholine, skin sensitivity, and PC20 allergen were collected retrospectively from 159 asthmatic patients participating in allergen challenge trials. Both the histamine and methacholine groups ( n=75 and n=84, respectively), were divided randomly into a reference group to derive new equations to predict PC20 allergen, and a validation group to test the new equations. Results Multiple linear regression analysis revealed that PC20 allergen could be predicted either from PC20 methacholine only (10log PC20 allergen=−0.902+0.741·10log PC20 methacholine) or from PC20 histamine and skin sensitivity (SS) (10log PC20 allergen=−0.494+0.231·10log SS+0.546·10log PC20 histamine). In the validation study, these new equations accurately predicted PC20 allergen following inhalation of HDM allergen allowing a safe starting concentration of allergen of three doubling concentrations below predicted PC20 allergen in all cases. Conclusion The early asthmatic response to inhaled HDM extract is predominantly determined by non-specific airway responsiveness to methacholine or histamine, whereas the influence of the cutaneous sensitivity to HDM appears to be rather limited. Our new equations accurately predict PC20 allergen and hence are suitable for implementation in HDM inhalation studies. [ABSTRACT FROM AUTHOR]

Adhesion molecule very late antigen-4 (VLA-4) is implicated in the recruitment and activation of inflammatory cells in asthma, including eosinophils, T cells and mast cells. VLA-4 antagonists have been proposed as a new anti-inflammatory treatment modality for asthma. Therefore, we investigated whether a single inhaled dose of VLA-4 antagonist GW559090X could protect against allergen-induced changes in airway responses and airway inflammation in patients with asthma. We performed a randomized, double-blind, three-way crossover study with single inhaled doses of 3 mg of GW559090X, 500 μg of fluticasone propionate (FP) or placebo in 15 patients with mild intermittent asthma, controlled with short-acting β2-agonists only. All patients developed a late asthmatic response (LAR) after allergen inhalation during screening. Study medication was administered 30 min prior to allergen challenge. Pre-dose and 24 h post-dose PC20 methacholine and levels of exhaled nitric oxide (eNO) were determined. At the given dose, VLA-4 antagonist GW559090X did not attenuate the early asthmatic response (EAR) when compared with placebo: mean AUC0(± SEM) (%fall h): 27.2 ± 3.7 and 21.9 ± 3.0 respectively ( P = 0.33); nor the LAR: mean AUC3-8h( ± SEM) (%fall h): 98.8 ± 12.9 and 94.8 ± 6.8 respectively ( P = 0.84). However, pretreatment with FP did attenuate both EAR and LAR when compared with placebo: mean AUC0-2h 11.6 ± 3.3 ( P = 0.024) and mean AUC3-8h 6.3 ± 7.6 ( P < 0.001). None of these treatments had an effect on allergen-induced changes in airway hyper-responsiveness or eNO levels. These findings suggest that VLA-4 may not play a major role in allergen-induced airway responses and inflammation in asthma. [ABSTRACT FROM AUTHOR]

Background Inflammatory and structural changes of the airway mucosa are chronic features of asthma. The mechanisms underlying these changes and their modulation by steroid prophylaxis have not been clarified. Objective We postulated that asymptomatic ongoing allergen exposure could drive airway inflammation as well as changes in the extracellular matrix (ECM), and that inhaled steroids could prevent this. Methods Therefore, we exposed patients with mild asthma to 2 weeks of repeated low-dose allergen, with concomitant inhaled steroid or placebo treatment. Bronchial biopsies, which were taken before and after this exposure, were stained and digitally analysed. The ECM proteins in asthmatics were also compared with a normal control group. Results Low-dose allergen exposure alone resulted in a significant increase of bronchial epithelial macrophages. Despite ongoing allergen exposure, inhaled steroids reduced the numbers of mucosal eosinophils, neutrophils and T lymphocytes. At baseline, the mean density of the proteoglycans (PGS) biglycan and decorin were, respectively, higher and lower in the bronchial mucosa of asthmatics as compared with normal controls. Steroid treatment, during allergen exposure, increased the mean density of the PGS biglycan and versican. Conclusion We conclude that chronic allergen exposure induces inflammatory changes in the bronchial mucosa. Despite ongoing allergen exposure, steroid treatment decreases mucosal inflammatory cells while altering PG density. The latter observation highlights the need to examine steroid-induced changes closely in the airway structure in patients with asthma. [ABSTRACT FROM AUTHOR]

Very late antigen-4 (VLA4) plays a key role in the recruitment of eosinophils in allergic responses in animal studies. We investigated whether pretreatment with multiple doses of a VLA4 receptor antagonist, HMR 1031, protects against allergen-induced airway responses and airway inflammation in humans. Fourteen asthmatics (7F/7M), 18–49 years, PC20 forced expiratory volume in 1 s (FEV1) methacholine (M) (<8 mg/mL; FEV1 82.3–116.1% predicted) with dual responses to inhaled allergen participated in a double-blind, placebo-controlled, cross-over study. Each treatment period consisted of 9 days, separated by ⩾2 weeks. Exhaled nitric oxide (eNO), PC20FEV1(M) and hypertonic saline-induced sputum was obtained on Days 1, 7 and 9. Subjects inhaled HMR 1031 (20 mg b.i.d.) or placebo (P) on Days 1–8. On Day 8, an allergen bronchoprovocation test was performed, the airway response was measured by FEV1, and expressed as %fall from baseline. Data from 12 evaluable subjects are presented here. Both treatments were well tolerated. There was no significant difference between HMR 1031 and P in the early asthamatic response: mean AUC (0–3 h)±SEM (%fall h): 26.01±4.26 and 17.41±4.26, respectively ( P=0.18), nor in the late response: mean AUC (3–9 h)±SEM (%fall h): 97.09±8.63 and 97.61±8.63, respectively, P=0.97. This corresponded to the absence of significant allergen-induced changes in PC20FEV1(M), eNO, sputum eosinophils and soluble inflammation markers between both treatment periods. Treatment with multiple inhaled doses of the VLA4 antagonist, HMR 1031, did not result in detectable protection against allergen-induced airway responses or airway inflammation in asthma. [ABSTRACT FROM AUTHOR]

Some patients with severe asthma cannot be controlled with high doses of inhaled steroids (ICS), which may be related to ongoing environmental allergen exposure. We investigated whether 10 weeks of high altitude allergen avoidance leads to sustained benefits regarding clinical and inflammatory markers of disease control in adolescents with persistent asthma despite treatment with high dose ICS. Eighteen atopic asthmatic adolescents (12–18 yr, 500–2000 µg ICS daily) with established house dust mite allergy, participated in a parallel-group study. Quality of life (PAQL), lung function, bronchial hyperresponsiveness (BHR) to adenosine and histamine, induced sputum and urine samples were collected repeatedly from 10 patients during a 10-week admission period to the Swiss Alps (alt. 1560 m) and at 6 weeks after return to sea level. Results were compared with those in eight patients, studied in their home environment at sea level for a similar time period. Throughout the study, asthma medication remained unchanged in both groups. During admission to high altitude, PAQL, lung function, BHR to adenosine and histamine, and urinary levels of eosinophil protein X (U-EPX), leukotriene E4 (U-LTE4) and 9α11β prostaglandin F2 (U-9α11β PGF2) improved significantly (P < 0.05), with a similar tendency for sputum eosinophils (P < 0.07). Furthermore, the changes in PAQL and BHR to adenosine and histamine were greater in the altitude than in the control group (P < 0.05). At 6 weeks after renewed allergen exposure at sea level, the improvements in PAQL (P < 0.05), BHR to adenosine (P < 0.07) and histamine (P < 0.05), as well as U-EPX (P < 0.05) and U-LTE4 (P < 0.05) were maintained. A short period of high altitude allergen avoidance, on top of regular treatment with ICS and long-acting β2-agonists, results in improvement of asthma, as assessed by clinical and inflammatory... [ABSTRACT FROM AUTHOR]

Explores whether airway hyperresponsiveness is an appropriate tool to define asthma phenotype. Pathophysiology of airway hyperresponsiveness; Segregation and linkage analysis in family asthma studies; Predictive value of airway hyperresponsiveness for the presence of asthma.

Background Induced sputum potentially allows monitoring of airway inflammation in patients with asthma in a non-invasive way. However, the relationship between the cellular content in sputum and airway tissue has not been fully clarified. Objective We compared the cellular compositions of hypertonic saline-induced sputum, bronchoalveolar lavage fluid (BAL) and bronchial biopsies in 18 clinically stable patients with mild to moderate atopic asthma (baseline FEV1: range 61-114%pred, PC20 methacholine: 0.04-4.7 mg/mL). They were treated with inhaled short-acting bronchodilators on demand, with (n =8) or without (n = 10) regular inhaled steroids. Methods Each patient underwent sputum induction and fiberoptic bronchoscopy on separate days in random order. Differential cell counts of induced sputum, bronchoalveolar lavage and bronchial wash were determined on May-Grünwald-Giemsa stained cytospins. Flow cytometry was performed on sputum and BAL samples. Immunohistochemical techniques were used to stain inflammatory cells in 6 micro;m cryostat sections of bronchial biopsies. Results Sputum cell differentials were not different between the patients with and without inhaled steroids, and showed a median value of 19.4% squamous cells, with 1.0% eosinophils, 3.3% lymphocytes, 28.7% neutrophils, 49.4% macrophages and 6.9% cylindric epithelial cells (in percentage non-squamous cells). The percentage eosinophils in sputum was significantly correlated with their percentage in bronchial wash (Rs = 0.52, P = 0.03) and in BAL (Rs = 0.55, P = 0.02), whilst there was a trend towards such a correlation between the number of eosinophils/mL sputum and the number of EG2+ eosinophils/mm² lamina propria in bronchial biopsies (Rs = 0.44, P = 0.07). In addition, the percentage of CD4+ lymphocytes correlated between sputum and BAL (Rs = 0.55, P = 0.03). Conclusion We conclude that the eosinophil counts in hypertonic saline-induced sputum from patients with asthma are related to those in bronchial wash and BAL and, to a lesser extent, with the counts in bronchial biopsies. This suggests that induced sputum can be used to monitor the presence and severity of airway inflammation in asthma. [ABSTRACT FROM AUTHOR]

Back ground Asthma exacerbations are closely associated with respiratory virus infections. However, the pathophysiological consequences of such infections in asthma arc largely unclear. Objective To examine the effect ot rhinovirus 16 (RV 16) infection on airway hypersensitivity to histamine, and on interleukin-8 (IL-8) in nasal lavage. Methods Twenty-seven non smoking atopic. mildly asthmatic subjects participated in a placebo-controlled. parallel study. A dose of 0.5-2.9 x 104 TCID50 RV 16 or placebo was nasally administered. Cold symptoms were recorded by questionnaire throughout the study. Histamine challenges were performed at entry, and on days 4 and 11 after inoculation. Nasal lavages were obtained at entry, and on days 2 and 9. The response to histamine was measured by PC20 (changes expressed as doubling doses: DD). IL-8 levels were obtained by ELISA, and were expressed in ng/ml. Results RV infection was confirmed by culture of nasal lavage and/or by antibody titre rise in each of the RVI6-treated subjects. Among the 19 RV16-treated subjects, eight developed severe cold symptoms. Baseline FEV1 did not change significantly during the study in either treatment group (P = 0.99). However, in the RV 16-treated subjects there was a decrease in PC20 at day 4, which was most pronounced in those with a severe cold (mean change ± SEM: -1.14 ± 0.28 DD. P=0.01). In addition. IL-8 levels increased in the RV 16 group at days 2 and 9 (P <0.001). The increase in nasal IL-8 at day 2 correlated significantly with the change in PC20 at day 4 (r = -0.48. P=0.04). Conclusion We conclude that the severity of cold, as induced by experimental RV16 infection, is a determinant of the increase in airway hypersensitivity to histamine in patients with asthma. Our results suggest that this may he mediated by an inflammatory mechanism, involving the release of chemokines such as IL-8. [ABSTRACT FROM AUTHOR]

Background Neuropeptides are likely to be implicated in the pathophysiology of allergen-induced airway responses. However, upon release in the airways, neuropeptides are potentially inactivated by neutral endopeptidase (NEP). Objective We hypothesized that NEP-inhibition by inhaled thiorphan (TH) would increase allergen-induced early (EAR) and late (LAR) asthmatic responses, and allergen-induced airway hyperresponsiveness to histamine in asthmatic subjects in vivo. The dose and dosing intervals of TH were derived from previous pharmacokinetic and dose-finding studies. Methods Nine non-smoking, atopic, asthmatic men with dual asthmatic responses to inhaled house-dust mite extract participated in a double-blind, placebo-controlled, cross-over study. During each study period PC20 histamine was measured 24 h before, and 3 and 24 h post-allergen. TH (1.25 mg/mL, 0.5 mL) or placebo (P) were aerosolized pre-allergen, and three times at 2 h intervals post-allergen (total dose of TH; 2.5 mg). Forced expiratory volume in one second (FEV1) was recorded and expressed as percentage fall from baseline. The EAR (0–3h) and the LAR (3–8 h) were defined as maximum percentage fail from the pre-allergen baseline and as corresponding areas under the time-response curves (AUC). Results As compared with P. TH failed to induce an acute effect on FEV1 at any of the timepoints (P > 0.08). There was no significant difference between P and TH in the EAR and the LAR; neither in terms of maximum percentage fall from baseline (mean ± SEM: EAR: 22.3±4.7% (P) and 20.4±4.1% (TH), P=0.75; LAR: 25.2±4.7% (P) and 26.4±5.8% (TH), P=0.77) nor in terms of AUC (P = 0.76). Correspondingly, the changes in PC20 histamine were not different between the two treatments (P>0.40). Conclusion We conclude that four adequate doses of the inhaled NEP-inhibitor, thiorphan, failed to potentiate allergen-induced airway responses in asthma. These results suggest that either neuropeptides do not play a predominant role in allergen-induced airway responses, or that allergen challenge induces NEP-dysfunction in humans in vivo. [ABSTRACT FROM AUTHOR]

Airway hyper responsiveness in asthma is characterized by increased airway sensitivity and by excessive maximal airway narrowing. Long-term inhalation therapy with nedocromil sodium has been shown to reduce increased airway sensitivity in asthma. However, it is unknown whether it also attenuates excessive airway narrowing. We studied the long-term effects of nedocromil on the maximal degree of airway narrowing to methacholine. Twenty-seven atopic asthmatic adults (21 39 years). with a measurable maximal-response plateau on the dose-response curve (20-55% fall in FEV1). were randomly allocated into two parallel treatment groups. They received either inhaled nedocromil 4 mg q.i.d. or placebo. for 8 weeks following a 2 weck baseline period. Every 2 weeks, complete dose-response curves to inhaled methacholine were obtained. The response was measured by FEV1 and by volume history standardized partial expiratory flow-volume curves (V40. A maximal-response plateau was considered if three or more of the highest data points fell within a 5% response range. the maximal response being the average value on the plateau (MFEV1, MV40p). Airway sensitivity was defined as the provocative concentration of methacholine causing a 20% fall in FEV1 (PC20FEV1) or 40% fall in V40p (PC40, V40p). Twenty-four subjects completed the study. Baseline FEV1 or V40p, did not change during either treatment (P>0.07). There were no significant changes in M FEV1 or MV40p during treatment with neclocromil (P> 0.07). Neither were these changes significantly different between the two groups (P>0.25). During treatment with nedocromil there was an increase in P20FEV1 and PC40p, which was maximal at 6 weeks (mean changed ± s.d.; 10±0.9 doubling dose (DD), and 0.8±0.7 DD, respectively, P=0.002). The changes in PC40 and PC40 were significantly different between the nedocromil and the placebo group (P<0.05). We conclude that long-term treatment with nedocromil, in spite of reducing airway sensitivity, does not reduce the maximal degree of airway narrowing to methacholine in mild to moderate asthmatics. This suggests that nedocromil may not be sufficient in the prevention of excessive airway narrowing in asthma. [ABSTRACT FROM AUTHOR]

The cover image, by P. Brinkman et al., is based on the Original Article Exhaled breath profiles in the monitoring of loss of control and clinical recovery in asthma, DOI: 10.1111/cea.12965. [ABSTRACT FROM AUTHOR]

Background: Asthma is a heterogeneous disease and its different phenotypes need to be better characterized from a biochemical-inflammatory standpoint. This study aimed to apply the metabolomic approach to exhaled breath condensate (breathomics) to discriminate different asthma phenotypes, with a particular focus on severe asthma in children., Methods: In this cross-sectional study, we recruited 42 asthmatic children (age, 8-17 years): 31 with nonsevere asthma (treated with inhaled steroids or not) and 11 with severe asthma. Fifteen healthy children were enrolled as controls. Children performed exhaled nitric oxide measurement, spirometry, exhaled breath condensate (EBC) collection. Condensate samples were analyzed using a metabolomic approach based on mass spectrometry., Results: A robust Bidirectional-Orthogonal Projections to Latent Structures-Discriminant Analysis (O2PLS-DA) model was found for discriminating both between severe asthma cases and healthy controls (R(2)  = 0.93; Q(2)  = 0.75) and between severe asthma and nonsevere asthma (R(2)  = 0.84; Q(2)  = 0.47). The metabolomic data analysis leads to a robust model also when the 3 groups of children were considered altogether (K = 0.80), indicating that each group is characterized by a specific metabolomic profile. Compounds related to retinoic acid, adenosine and vitamin D (Human Metabolome Database) were relevant for the discrimination between groups., Conclusion: The metabolomic profiling of EBC could clearly distinguish different biochemical-metabolic profiles in asthmatic children and enabled the severe asthma phenotype to be fully discriminated. The breathomics approach may therefore be suitable for discriminating between different asthma metabolic phenotypes., (© 2012 John Wiley & Sons A/S.)