Patients with severe refractory asthma pose a major healthcare problem. Over the last decade it has become clear that, new targeted treatments are needed for severe refractory asthma. To develop these treatments further characterisation and classification of people with asthma is needed. This paper presents the U-BIOPRED international consensus on the definition and diagnosis of severe asthma in both adults and children. The consensus will be used for the selection of patients for the U-BIOPRED study. It outlines the differences between ‘problematic’, ‘difficult’ and ‘severe refractory’ asthma, and provides a systematic approach to evaluating patients with chronic severe asthma symptoms for use in clinical research and practice.
Cellular and molecular pathways in severe asthma are highly complex and relatively unknown. Increased understanding may be obtained by transcriptomic analysis of endobronchial biopsies. We hypothesised that the transcriptomic profile of whole human endobronchial biopsies differs between asthma patients of different severities and controls.
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RNA viruses are a major cause of respiratory infections and are known to exacerbate asthma and other respiratory diseases. Our aim was to test the ability of poly(I:C) (polyinosinic:polycytidylic acid), a viral surrogate, to elicit exacerbation in a model of severe asthma driven by HDM (house dust mite) in FCA (Freund's complete adjuvant).
Poly(I:C) was administered intranasally around the HDM challenge in FCA-HDM-sensitized animals. Changes in AHR (airway hyperresponsiveness), BALF (bronchoalveolar lavage fluid) inflammatory infiltrate, HDM-specific immunoglobulins and cytokine/chemokine release were evaluated at different points after the challenge.
We have set up a model that mimics key aspects of viral exacerbation in a corticosteroid-refractory asthmatic phenotype which could be used to evaluate new therapies for this condition.
Current research tells us that electronic nose technology is most frequently used in breath analysis. In this study a number of different devices were used to test the reliability and usefulness of e-nose technology in clinical practice settings. The tests have showed that e-nose technology can detect chemicals at concentration levels of around 1 part per million and found that detection of down to tens of parts per billion is possible. This study has been able to map the performance of different devices, and provides the first step toward quality assurance of e-nose data in the medical setting.
To newly discover severe asthma phenotypes by unbiased cluster analysis based on metabolomic fingerprints from exhaled breath by the U-BIOPRED composite electronic nose (eNose) platform.
Oxidative stress is believed to be a major driver of inflammation in smoking asthmatics. The U-BIOPRED project recruited a cohort of Severe Asthma smokers/ex-smokers (SAs/ex) and non-smokers (SAn) with extensive clinical and biomarker information enabling characterization of these subjects. We investigated oxidative stress in severe asthma subjects by analysing urinary 8-iso-PGF2α and the mRNA-expression of the main pro-oxidant (NOX2; NOSs) and anti-oxidant (SODs; CAT; GPX1) enzymes in the airways of SAs/ex and SAn. All the severe asthma U-BIOPRED subjects were further divided into current smokers with severe asthma (CSA), ex-smokers with severe asthma (ESA) and non-smokers with severe asthma (NSA) to deepen the effect of active smoking. Clinical data, urine and sputum were obtained from severe asthma subjects. A bronchoscopy to obtain bronchial biopsy and brushing was performed in a subset of subjects. The main clinical data were analysed for each subset of subjects (urine-8-iso-PGF2α; IS-transcriptomics; BB-transcriptomics; BBr-transcriptomics). Urinary 8-iso-PGF2α was quantified using mass spectrometry. Sputum, bronchial biopsy and bronchial brushing were processed for mRNA expression microarray analysis. Urinary 8-iso-PGF2α was increased in SAs/ex, median (IQR) = 31.7 (24.5-44.7) ng/mmol creatinine, compared to SAn, median (IQR) = 26.6 (19.6-36.6) ng/mmol creatinine (p< 0.001), and in CSA, median (IQR) = 34.25 (24.4-47.7), vs. ESA, median (IQR) = 29.4 (22.3-40.5), and NSA, median (IQR) = 26.5 (19.6-16.6) ng/mmol creatinine (p = 0.004). Sputum mRNA expression of NOX2 was increased in SAs/ex compared to SAn (probe sets 203922_PM_s_at fold-change = 1.05 p = 0.006; 203923_PM_s_at fold-change = 1.06, p = 0.003; 233538_PM_s_at fold-change = 1.06, p = 0.014). The mRNA expression of antioxidant enzymes were similar between the two severe asthma cohorts in all airway samples. NOS2 mRNA expression was decreased in bronchial brushing of SAs/ex compared to SAn (fold-change = -1.10; p = 0.029). NOS2 mRNA expression in bronchial brushing correlated with FeNO (Kendal's Tau = 0.535; p< 0.001). From clinical and inflammatory analysis, FeNO was lower in CSA than in ESA in all the analysed subject subsets (p< 0.01) indicating an effect of active smoking. Results about FeNO suggest its clinical limitation, as inflammation biomarker, in severe asthma active smokers. These data provide evidence of greater systemic oxidative stress in severe asthma smokers as reflected by a significant changes of NOX2 mRNA expression in the airways, together with elevated urinary 8-iso-PGF2α in the smokers/ex-smokers group. Trial registration ClinicalTrials.gov-Identifier: NCT01976767.
Gastro-oesophageal reflux disease (GORD) and obesity are associated with frequent exacerbations and poor quality of life in asthmatics. Multiple mechanisms have been proposed for the effect of obesity, including modification of inflammation affecting epithelial cell proliferation and wound repair, while the role of GORD is poorly understood and proton pump inhibitor (PPI) are of variable efficacy. GORD might exert a deleterious effect by inducing vagal reflex, neuroinflammation and directly (via microaspiration) triggering airway inflammation. Studies of reflux in animal models and human bronchial epithelial cell culture show varying impact on inflammation and airway remodelling.
U-BIOPRED has added substantially to both our clinical and mechanistic understanding of asthma. This ERS Monograph chapter provides an overview of the project and considers the organisational lessons that can be learnt from U-BIOPRED: the importance of a flexible structure of governance, use of adequate data management resources, and a focus on the depth of patient characterisation rather than cohort size. With the right effort, multiple stakeholders, including patients, can work together in a true collaborative spirit.
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.
Eosinophilic inflammation in asthma is predictive for responses to inhaled steroids. The application of sputum analysis is limited by requirements of lab facilities and not-directly available results. Exhaled air metabolomic profiles are associated with eosinophilic inflammation in asthma (Ibrahim et al. Thorax 2011; 66(9): 804-9). We hypothesized that breathprint profiles analysed by electronic noses (eNoses) can be surrogate markers for eosinophilic airway inflammation.
This commentary talks about patient involvement in one of the biggest EU projects to date—U-BIOPRED. It describes how people and carers of people with asthma have been able to develop and drive their input and have their voice heard among the >200 healthcare professional project members.
Five key principles for the success of the patient involvement group are presented: involve early, involve deeply, have patients feedback on project progress, include patients in dissemination and help patients convey their own story.
This group has been used as an example for other EU-funded projects, and the patient involvement group will be maintained after the end of the project to ensure that their experience and knowledge can help develop best practice in the future.
Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality and morbidity worldwide, which is increasing particularly among females. Smoking represents the main risk factor for developing COPD; however, the chronic inflammation persists following smoking cessation. A plethora of inflammatory mediators are likely to orchestrate this persistent inflammation with resulting tissue remodeling and decline in lung function. We have investigated the effects of smoking, in relation to disease, on oxylipins as key lipid mediators in the inflammatory response in the lower airways.
The U-BIOPRED consortium is an EU-wide collective of academics, pharmaceutical companies and other organisations working collaboratively to better understand severe asthma. The U-BIOPRED proteomics platform has been developed to discover novel protein biomarkers to enable disease stratification, and to enhance our molecular understanding of this disease. Using innovative sample processing, mass spectrometry and data-mining methodologies, we report here the most comprehensive quantitative proteomic analysis of human induced sputum to date.
Severe asthma is a heterogeneous condition, as shown by independent cluster analyses based on demographic, clinical, and inflammatory characteristics. A next step is to identify molecularly driven phenotypes using "omics" technologies. Molecular fingerprints of exhaled breath are associated with inflammation and can qualify as noninvasive assessment of severe asthma phenotypes.OBJECTIVES:
We aimed (1) to identify severe asthma phenotypes using exhaled metabolomic fingerprints obtained from a composite of electronic noses (eNoses) and (2) to assess the stability of eNose-derived phenotypes in relation to within-patient clinical and inflammatory changes.METHODS:
In this longitudinal multicenter study exhaled breath samples were taken from an unselected subset of adults with severe asthma from the U-BIOPRED cohort. Exhaled metabolites were analyzed centrally by using an assembly of eNoses. Unsupervised Ward clustering enhanced by similarity profile analysis together with K-means clustering was performed. For internal validation, partitioning around medoids and topological data analysis were applied. Samples at 12 to 18 months of prospective follow-up were used to assess longitudinal within-patient stability.RESULTS:
Data were available for 78 subjects (age, 55 years [interquartile range, 45-64 years]; 41% male). Three eNose-driven clusters (n = 26/33/19) were revealed, showing differences in circulating eosinophil (P = .045) and neutrophil (P = .017) percentages and ratios of patients using oral corticosteroids (P = .035). Longitudinal within-patient cluster stability was associated with changes in sputum eosinophil percentages (P = .045).CONCLUSIONS:
We have identified and followed up exhaled molecular phenotypes of severe asthma, which were associated with changing inflammatory profile and oral steroid use. This suggests that breath analysis can contribute to the management of severe asthma.
Preventing exacerbations of asthma is a major goal in current guidelines. We aimed to develop a prediction model enabling practitioners to identify patients at risk of severe exacerbations who could potentially benefit from a change in management.METHODS:
We used data from a 12-month primary care pragmatic trial; candidate predictors were identified from GINA 2014 and selected with a multivariable bootstrapping procedure. Three models were constructed, based on: (1) history, (2) history+spirometry and (3) history+spirometry+FeNO. Final models were corrected for overoptimism by shrinking the regression coefficients; predictive performance was assessed by the area under the receiver operating characteristic curve (AUROC) and Hosmer-Lemeshow test. Models were externally validated in a data set including patients with severe asthma (Unbiased BIOmarkers in PREDiction of respiratory disease outcomes).RESULTS:
80/611 (13.1%) participants experienced ≥1 severe exacerbation. Five predictors (Asthma Control Questionnaire score, current smoking, chronic sinusitis, previous hospital admission for asthma and ≥1 severe exacerbation in the previous year) were retained in the history model (AUROC 0.77 (95% CI 0.75 to 0.80); Hosmer-Lemeshow p value 0.35). Adding spirometry and FeNO subsequently improved discrimination slightly (AUROC 0.79 (95% CI 0.77 to 0.81) and 0.80 (95% CI 0.78 to 0.81), respectively). External validation yielded AUROCs of 0.72 (95% CI 0.70 to 0.73; 71 to 0.74 and 0.71 to 0.73) for the three models, respectively; calibration was best for the spirometry model.CONCLUSIONS:
A simple history-based model extended with spirometry identifies patients who are prone to asthma exacerbations. The additional value of FeNO is modest. These models merit an implementation study in clinical practice to assess their utility.TRIAL REGISTRATION NUMBER:
The role of IL-17 immunity is well established in patients with inflammatory diseases, such as psoriasis and inflammatory bowel disease, but not in asthmatic patients, in whom further study is required.OBJECTIVE:
We sought to undertake a deep phenotyping study of asthmatic patients with upregulated IL-17 immunity.METHODS:
Whole-genome transcriptomic analysis was performed by using epithelial brushings, bronchial biopsy specimens (91 asthmatic patients and 46 healthy control subjects), and whole blood samples (n = 498) from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. Gene signatures induced in vitro by IL-17 and IL-13 in bronchial epithelial cells were used to identify patients with IL-17-high and IL-13-high asthma phenotypes.RESULTS:
Twenty-two of 91 patients were identified with IL-17, and 9 patients were identified with IL-13 gene signatures. The patients with IL-17-high asthma were characterized by risk of frequent exacerbations, airway (sputum and mucosal) neutrophilia, decreased lung microbiota diversity, and urinary biomarker evidence of activation of the thromboxane B2 pathway. In pathway analysis the differentially expressed genes in patients with IL-17-high asthma were shared with those reported as altered in psoriasis lesions and included genes regulating epithelial barrier function and defense mechanisms, such as IL1B, IL6, IL8, and β-defensin.CONCLUSION:
The IL-17-high asthma phenotype, characterized by bronchial epithelial dysfunction and upregulated antimicrobial and inflammatory response, resembles the immunophenotype of psoriasis, including activation of the thromboxane B2 pathway, which should be considered a biomarker for this phenotype in further studies, including clinical trials targeting IL-17
Influenza virus triggers severe asthma exacerbations for which no adequate treatment is available. It is known that IL-33 levels correlate with exacerbation severity, but its role in the immunopathogenesis of exacerbations has remained elusive.OBJECTIVE:
We hypothesized that IL-33 is necessary to drive asthma exacerbations. We intervened with the IL-33 cascade and sought to dissect its role, also in synergy with thymic stromal lymphopoietin (TSLP), in airway inflammation, antiviral activity, and lung function. We aimed to unveil the major source of IL-33 in the airways and IL-33-dependent mechanisms that underlie severe asthma exacerbations.METHODS:
Patients with mild asthma were experimentally infected with rhinovirus. Mice were chronically exposed to house dust mite extract and then infected with influenza to resemble key features of exacerbations in human subjects. Interventions included the anti-IL-33 receptor ST2, anti-TSLP, or both.RESULTS:
We identified bronchial ciliated cells and type II alveolar cells as a major local source of IL-33 during virus-driven exacerbation in human subjects and mice, respectively. By blocking ST2, we demonstrated that IL-33 and not TSLP was necessary to drive exacerbations. IL-33 enhanced airway hyperresponsiveness and airway inflammation by suppressing innate and adaptive antiviral responses and by instructing epithelial cells and dendritic cells of house dust mite-sensitized mice to dampen IFN-β expression and prevent the TH1-promoting dendritic cell phenotype. IL-33 also boosted luminal NETosis and halted cytolytic antiviral activities but did not affect the TH2 response.CONCLUSION:
Interventions targeting the IL-33/ST2 axis could prove an effective acute short-term therapy for virus-induced asthma exacerbations.
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