Ariane H. Wagener

Diagnosis and definition of severe refractory asthma: an international consensus statement from the Innovative Medicine Initiative (IMI)

Patients with severe refractory asthma pose a major healthcare problem. Over the last decade it has become increasingly clear that, for the development of new targeted therapies, there is an urgent need for further characterisation and classification of these patients. The Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) consortium is a pan-European public-private collaboration funded by the European Commission Innovative Medicines Initiative of the European Union. U-BIOPRED aims to subphenotype patients with severe refractory asthma by using an innovative systems biology approach. This paper presents the U-BIOPRED international consensus on the definition and diagnosis of severe asthma, aligning the latest concepts in adults as well as in children. The consensus is based on existing recommendations up to 2010 and will be used for the selection of patients for the upcoming U-BIOPRED study. It includes the differentiation between ‘problematic’, ‘difficult’ and ‘severe refractory’ asthma, and provides a systematic algorithmic approach to the evaluation of patients presenting with chronic severe asthma symptoms for use in clinical research and specialised care.

External validation of blood eosinophils, FE NO and serum periostin as surrogates for sputum eosinophils in asthma

Background Monitoring sputum eosinophils in asthma predicts exacerbations and improves management of asthma. Thus far, blood eosinophils and FENO show contradictory results in predicting eosinophilic airway inflammation. More recently, serum periostin was proposed as a novel biomarker for eosinophilic inflammation. Objectives Quantifying the mutual relationships of blood eosinophils, FENO, and serum periostin with sputum eosinophils by external validation in two independent cohorts across various severities of asthma. Methods The first cohort consisted of 110 patients with mild to moderate asthma (external validation cohort). The replication cohort consisted of 37 patients with moderate to severe asthma. Both cohorts were evaluated cross-sectionally. Sputum was induced for the assessment of eosinophils. In parallel, blood eosinophil counts, serum periostin concentrations and FENO were assessed. The diagnostic accuracy of these markers to identify eosinophilic asthma (sputum eosinophils ≥3%) was calculated using receiver operating characteristics area under the curve (ROC AUC). Results In the external validation cohort, ROC AUC for blood eosinophils was 89% (p<0.001) and for FENO level 78% (p<0.001) to detect sputum eosinophilia ≥3%. Serum periostin was not able to distinguish eosinophilic from non-eosinophilic airway inflammation (ROC AUC=55%, p=0.44). When combining these three variables, no improvement was seen. The diagnostic value of blood eosinophils was confirmed in the replication cohort (ROC AUC 85%, p<0.001). Conclusions In patients with mild to moderate asthma, as well as patients with more severe asthma, blood eosinophils had the highest accuracy in the identification of sputum eosinophilia in asthma. The use of blood eosinophils can facilitate individualised treatment and management of asthma. Trial registration NTR1846 and NTR2364.

Clinical and inflammatory characteristics of the European U-BIOPRED adult severe asthma cohort

U-BIOPRED is a European Union consortium of 20 academic institutions, 11 pharmaceutical companies and six patient organisations with the objective of improving the understanding of asthma disease mechanisms using a systems biology approach. This cross-sectional assessment of adults with severe asthma, mild/moderate asthma and healthy controls from 11 European countries consisted of analyses of patient-reported outcomes, lung function, blood and airway inflammatory measurements. Patients with severe asthma (nonsmokers, n=311; smokers/ex-smokers, n=110) had more symptoms and exacerbations compared to patients with mild/moderate disease (n=88) (2.5 exacerbations versus 0.4 in the preceding 12 months; p<0.001), with worse quality of life, and higher levels of anxiety and depression. They also had a higher incidence of nasal polyps and gastro-oesophageal reflux with lower lung function. Sputum eosinophil count was higher in severe asthma compared to mild/moderate asthma (median count 2.99% versus 1.05%; p=0.004) despite treatment with higher doses of inhaled and/or oral corticosteroids. Consistent with other severe asthma cohorts, U-BIOPRED is characterised by poor symptom control, increased comorbidity and airway inflammation, despite high levels of treatment. It is well suited to identify asthma phenotypes using the array of “omic” datasets that are at the core of this systems medicine approach. Severe asthma results in more airway inflammation, worse symptoms and lower lung function, despite increased therapy http://ow.ly/QznR3

Electronic Nose Breathprints Are Independent of Acute Changes in Airway Caliber in Asthma

Molecular profiling of exhaled volatile organic compounds (VOC) by electronic nose technology provides breathprints that discriminate between patients with different inflammatory airway diseases, such as asthma and COPD. However, it is unknown whether this is determined by differences in airway caliber. We hypothesized that breathprints obtained by electronic nose are independent of acute changes in airway caliber in asthma. Ten patients with stable asthma underwent methacholine provocation (Visit 1) and sham challenge with isotonic saline (Visit 2). At Visit 1, exhaled air was repetitively collected pre-challenge, after reaching the provocative concentration (PC20) causing 20% fall in forced expiratory volume in 1 second (FEV1) and after subsequent salbutamol inhalation. At Visit 2, breath was collected pre-challenge, post-saline and post-salbutamol. At each occasion, an expiratory vital capacity was collected after 5 min of tidal breathing through an inspiratory VOC-filter in a Tedlar bag and sampled by electronic nose (Cyranose 320). Breathprints were analyzed with principal component analysis and individual factors were compared with mixed model analysis followed by pairwise comparisons. Inhalation of methacholine led to a 30.8 ± 3.3% fall in FEV1 and was followed by a significant change in breathprint (p = 0.04). Saline inhalation did not induce a significant change in FEV1, but altered the breathprint (p = 0.01). However, the breathprint obtained after the methacholine provocation was not significantly different from that after saline challenge (p = 0.27). The molecular profile of exhaled air in patients with asthma is altered by nebulized aerosols, but is not affected by acute changes in airway caliber. Our data demonstrate that breathprints by electronic nose are not confounded by the level of airway obstruction.

The Impact of Allergic Rhinitis and Asthma on Human Nasal and Bronchial Epithelial Gene Expression

Background The link between upper and lower airways in patients with both asthma and allergic rhinitis is still poorly understood. As the biological complexity of these disorders can be captured by gene expression profiling we hypothesized that the clinical expression of rhinitis and/or asthma is related to differential gene expression between upper and lower airways epithelium. Objective Defining gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles. Methods This cross-sectional study included 18 subjects (6 allergic asthma and allergic rhinitis; 6 allergic rhinitis; 6 healthy controls). The estimated false discovery rate comparing 6 subjects per group was approximately 5%. RNA was extracted from isolated and cultured epithelial cells from bronchial brushings and nasal biopsies, and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and Bioconductor Limma package. For gene ontology GeneSpring GX12 was used. Results The study was successfully completed by 17 subjects (6 allergic asthma and allergic rhinitis; 5 allergic rhinitis; 6 healthy controls). Using correction for multiple testing, 1988 genes were differentially expressed between healthy lower and upper airway epithelium, whereas in allergic rhinitis with or without asthma this was only 40 and 301 genes, respectively. Genes influenced by allergic rhinitis with or without asthma were linked to lung development, remodeling, regulation of peptidases and normal epithelial barrier functions. Conclusions Differences in epithelial gene expression between the upper and lower airway epithelium, as observed in healthy subjects, largely disappear in patients with allergic rhinitis with or without asthma, whilst new differences emerge. The present data identify several pathways and genes that might be potential targets for future drug development.

dsRNA-induced changes in gene expression profiles of primary nasal and bronchial epithelial cells from patients with asthma, rhinitis and controls

BackgroundRhinovirus infections are the most common cause of asthma exacerbations. The complex responses by airway epithelium to rhinovirus can be captured by gene expression profiling. We hypothesized that: a) upper and lower airway epithelium exhibit differential responses to double-stranded RNA (dsRNA), and b) that this is modulated by the presence of asthma and allergic rhinitis.ObjectivesIdentification of dsRNA-induced gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles.MethodsThis study had a cross-sectional design including 18 subjects: 6 patients with allergic asthma with concomitant rhinitis, 6 patients with allergic rhinitis, and 6 healthy controls. Comparing 6 subjects per group, the estimated false discovery rate was approximately 5%. RNA was extracted from isolated and cultured primary epithelial cells from nasal biopsies and bronchial brushings stimulated with dsRNA (poly(I:C)), and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and the Bioconductor Limma package. Overrepresentation of gene ontology groups were captured by GeneSpring GX12.ResultsIn total, 17 subjects completed the study successfully (6 allergic asthma with rhinitis, 5 allergic rhinitis, 6 healthy controls). dsRNA-stimulated upper and lower airway epithelium from asthma patients demonstrated significantly fewer induced genes, exhibiting reduced down-regulation of mitochondrial genes. The majority of genes related to viral responses appeared to be similarly induced in upper and lower airways in all groups. However, the induction of several interferon-related genes (IRF3, IFNAR1, IFNB1, IFNGR1, IL28B) was impaired in patients with asthma.ConclusionsdsRNA differentially changes transcriptional profiles of primary nasal and bronchial epithelial cells from patients with allergic rhinitis with or without asthma and controls. Our data suggest that respiratory viruses affect mitochondrial genes, and we identified disease-specific genes that provide potential targets for drug development.

Transcriptomic gene signatures associated with persistent airflow limitation in patients with severe asthma

A proportion of severe asthma patients suffers from persistent airflow limitation (PAL), often associated with more symptoms and exacerbations. Little is known about the underlying mechanisms. Here, our aim was to discover unexplored potential mechanisms using Gene Set Variation Analysis (GSVA), a sensitive technique that can detect underlying pathways in heterogeneous samples. Severe asthma patients from the U-BIOPRED cohort with PAL (post-bronchodilator forced expiratory volume in 1 s/forced vital capacity ratio below the lower limit of normal) were compared with those without PAL. Gene expression was assessed on the total RNA of sputum cells, nasal brushings, and endobronchial brushings and biopsies. GSVA was applied to identify differentially enriched predefined gene signatures based on all available gene expression publications and data on airways disease. Differentially enriched gene signatures were identified in nasal brushings (n=1), sputum (n=9), bronchial brushings (n=1) and bronchial biopsies (n=4) that were associated with response to inhaled steroids, eosinophils, interleukin-13, interferon-α, specific CD4+ T-cells and airway remodelling. PAL in severe asthma has distinguishable underlying gene networks that are associated with treatment, inflammatory pathways and airway remodelling. These findings point towards targets for the therapy of PAL in severe asthma. Persistent airflow limitation in severe asthma is associated with a mechanism involving IL-13 and remodelling http://ow.ly/JYcC30daSRf

An investigation on e-nose platform relevance to respiratory diseases

The e-nose platform here presented gather up 190 sensors belonging to four different e-nose technologies. This platform is used in the context of a European project (U-Biopred) measuring exhaled breath samples collected from patients affected by mild or severe asthma. Sensor responses are investigated asking which disease can be diagnosed on the basis of sensor correlation with specific clinical parameters.