Assessment of breath volatile organic compounds in acute cardiorespiratory breathlessness: a protocol describing a prospective real-world observational study

Introduction Patients presenting with acute undifferentiated breathlessness are commonly encountered in admissions units across the UK. Existing blood biomarkers have clinical utility in distinguishing patients with single organ pathologies but have poor discriminatory power in multifactorial presentations. Evaluation of volatile organic compounds (VOCs) in exhaled breath offers the potential to develop biomarkers of disease states that underpin acute cardiorespiratory breathlessness, owing to their proximity to the cardiorespiratory system. To date, there has been no systematic evaluation of VOC in acute cardiorespiratory breathlessness. The proposed study will seek to use both offline and online VOC technologies to evaluate the predictive value of VOC in identifying common conditions that present with acute cardiorespiratory breathlessness. Methods and analysis A prospective real-world observational study carried out across three acute admissions units within Leicestershire. Participants with self-reported acute breathlessness, with a confirmed primary diagnosis of either acute heart failure, community-acquired pneumonia and acute exacerbation of asthma or chronic obstructive pulmonary disease will be recruited within 24 hours of admission. Additionally, school-age children admitted with severe asthma will be evaluated. All participants will undergo breath sampling on admission and on recovery following discharge. A range of online technologies including: proton transfer reaction mass spectrometry, gas chromatography ion mobility spectrometry, atmospheric pressure chemical ionisation-mass spectrometry and offline technologies including gas chromatography mass spectroscopy and comprehensive two-dimensional gas chromatography-mass spectrometry will be used for VOC discovery and replication. For offline technologies, a standardised CE-marked breath sampling device (ReCIVA) will be used. All recruited participants will be characterised using existing blood biomarkers including C reactive protein, brain-derived natriuretic peptide, troponin-I and blood eosinophil levels and further evaluated using a range of standardised questionnaires, lung function testing, sputum cell counts and other diagnostic tests pertinent to acute disease. Ethics and dissemination The National Research Ethics Service Committee East Midlands has approved the study protocol (REC number: 16/LO/1747). Integrated Research Approval System (IRAS) 198921. Findings will be presented at academic conferences and published in peer-reviewed scientific journals. Dissemination will be facilitated via a partnership with the East Midlands Academic Health Sciences Network and via interaction with all UK-funded Medical Research Council and Engineering and Physical Sciences Research Council molecular pathology nodes. Trial registration number NCT0367299

Airway Smooth Muscle Hypercontractility in Asthma

In recent years, asthma has been defined primarily as an inflammatory disorder with emphasis on inflammation being the principle underlying pathophysiological characteristic driving airway obstruction and remodelling. Morphological abnormalities of asthmatic airway smooth muscle (ASM), the primary structure responsible for airway obstruction seen in asthma, have long been described, but surprisingly, until recently, relatively small number of studies investigated whether asthmatic ASM was also fundamentally different in its functional properties. Evidence from recent studies done on single ASM cells and on ASM impregnated gel cultures have shown that asthmatic ASM is intrinsically hypercontractile. Several elements of the ASM contraction apparatus in asthmatics and in animal models of asthma have been found to be different from nonasthmatics. These differences include some regulatory contractile proteins and also some components of both the calcium-dependent and calcium-independent contraction signalling pathways. Furthermore, oxidative stress was also found to be heightened in asthmatic ASM and contributes to hypercontractility. Understanding the abnormalities and mechanisms driving asthmatic ASM hypercontractility provides a great potential for the development of new targeted drugs, other than the conventional current anti-inflammatory and bronchodilator therapies, to address the desperate unmet need especially in patients with severe and persistent asthma

D prostanoid receptor 2 (chemoattractant receptor-homologous molecule expressed on TH2 cells) protein expression in asthmatic patients and its effects on bronchial epithelial cells.

BACKGROUND: The D prostanoid receptor 2 (DP2; also known as chemoattractant receptor-homologous molecule expressed on TH2 cells) is implicated in the pathogenesis of asthma, but its expression within bronchial biopsy specimens is unknown. OBJECTIVES: We sought to investigate the bronchial submucosal DP2 expression in asthmatic patients and healthy control subjects and to explore its functional role in epithelial cells. METHODS: DP2 protein expression was assessed in bronchial biopsy specimens from asthmatic patients (n = 22) and healthy control subjects (n = 10) by using immunohistochemistry and in primary epithelial cells by using flow cytometry, immunofluorescence, and quantitative RT-PCR. The effects of the selective DP2 agonist 13, 14-dihydro-15-keto prostaglandin D2 on epithelial cell migration and differentiation were determined. RESULTS: Numbers of submucosal DP2(+) cells were increased in asthmatic patients compared with those in healthy control subjects (mean [SEM]: 78 [5] vs 22 [3]/mm(2) submucosa, P < .001). The bronchial epithelium expressed DP2, but its expression was decreased in asthmatic patients compared with that seen in healthy control subjects (mean [SEM]: 21 [3] vs 72 [11]/10 mm(2) epithelial area, P = .001), with similar differences observed in vitro by primary epithelial cells. Squamous metaplasia of the bronchial epithelium was increased in asthmatic patients and related to decreased DP2 expression (rs = 0.69, P < .001). 13, 14-Dihydro-15-keto prostaglandin D2 promoted epithelial cell migration and at air-liquid interface cultures increased the number of MUC5AC(+) and involucrin-positive cells, which were blocked with the DP2-selective antagonist AZD6430. CONCLUSIONS: DP2 is expressed by the bronchial epithelium, and its activation drives epithelial differentiation, suggesting that in addition to its well-characterized role in inflammatory cell migration, DP2 might contribute to airway remodeling in asthmatic patients

Computational modeling of the obstructive lung diseases asthma and COPD

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by airway obstruction and airflow imitation and pose a huge burden to society. These obstructive lung diseases impact the lung physiology across multiple biological scales. Environmental stimuli are introduced via inhalation at the organ scale, and consequently impact upon the tissue, cellular and sub-cellular scale by triggering signaling pathways. These changes are propagated upwards to the organ level again and vice versa. In order to understand the pathophysiology behind these diseases we need to integrate and understand changes occurring across these scales and this is the driving force for multiscale computational modeling. There is an urgent need for improved diagnosis and assessment of obstructive lung diseases. Standard clinical measures are based on global function tests which ignore the highly heterogeneous regional changes that are characteristic of obstructive lung disease pathophysiology. Advances in scanning technology such as hyperpolarized gas MRI has led to new regional measurements of ventilation, perfusion and gas diffusion in the lungs, while new image processing techniques allow these measures to be combined with information from structural imaging such as Computed Tomography (CT). However, it is not yet known how to derive clinical measures for obstructive diseases from this wealth of new data. Computational modeling offers a powerful approach for investigating this relationship between imaging measurements and disease severity, and understanding the effects of different disease subtypes, which is key to developing improved diagnostic methods. Gaining an understanding of a system as complex as the respiratory system is difficult if not impossible via experimental methods alone. Computational models offer a complementary method to unravel the structure-function relationships occurring within a multiscale, multiphysics system such as this. Here we review the currentstate-of-the-art in techniques developed for pulmonary image analysis, development of structural models of therespiratory system and predictions of function within these models. We discuss application of modeling techniques to obstructive lung diseases, namely asthma and emphysema and the use of models to predict response to therapy. Finally we introduce a large European project, AirPROM that is developing multiscale models toinvestigate structure-function relationships in asthma and COPD

Effect of levofloxacin on neutrophilic airway inflammation in stable COPD: a randomized, double-blind, placebo-controlled trial

RATIONALE: Airway inflammation persists after smoking cessation in established chronic obstructive pulmonary disease (COPD), suggesting that other factors drive the airway inflammatory response. OBJECTIVES: We tested the hypothesis that high levels of bacterial colonization are associated with increased levels of neutrophilic airway inflammation in stable COPD by examining the cross-sectional relationship between these measurements and by conducting a randomized, double-blind, placebo-controlled study of the effect of levofloxacin in patients with stable COPD. METHODS: Patients were randomized to receive either levofloxacin 500 mg daily or placebo for 7 days and underwent sputum induction for a differential cell count and quantitative bacterial analysis at baseline and at days 7, 14, and 28. RESULTS: Sputum percentage neutrophil count correlated with airway bacterial load at baseline (r=0.56; P=0.003). Levofloxacin reduced bacterial load compared with placebo by 4.9-fold (95% confidence interval, 1.4-25.7; P=0.02) at day 7 but had no effect at any point on any marker of neutrophilic airway inflammation. In patients with a baseline bacterial load of more than 10(6) cfu/mL, levofloxacin treatment was associated with a 26.5% (95% confidence interval, 1.8%-51.3%; P=0.04) greater reduction in the percentage neutrophil count compared with placebo at day 7. Change in percentage neutrophil count correlated significantly with baseline airway bacterial load and change in airway bacterial load. CONCLUSION: In stable COPD, levofloxacin treatment causes a short-term reduction in bacterial load. This is associated with a reduction in neutrophilic airway inflammation in patients with high bacterial loads. Further studies are required to investigate whether this effect is clinically advantageous

Human Group 2 innate lymphoid cells do not express the IL-5 receptor

Group 2 innate lymphoid cells (ILC2s) are upstream regulators of IL-5-dependent eosinophil function in asthma. We’ve shown that ILC2s do not express the IL-5R and thus IL-5R- dependent therapeutic interventions (e.g. Benralizumab) are unlikely to be mediated directly on ILC2s

[Ca2+]i oscillations in ASM: relationship with persistent airflow obstruction in asthma.

The cause of airway smooth muscle (ASM) hypercontractility in asthma is not fully understood. The relationship of spontaneous intracellular calcium oscillation frequency in ASM to asthma severity was investigated. Oscillations were increased in subjects with impaired lung function abolished by extracellular calcium removal, attenuated by caffeine and unaffected by verapamil or nitrendipine. Whether modulation of increased spontaneous intracellular calcium oscillations in ASM from patients with impaired lung function represents a therapeutic target warrants further investigation

CCL2 release by airway smooth muscle is increased in asthma and promotes fibrocyte migration.

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Airway smooth muscle-derived CCL2 mediates FC migration and potentially contributes to the development of ASM hyperplasia in asthma

Investigating the role of pentraxin 3 as a biomarker for bacterial infection in subjects with COPD

BACKGROUND: Pentraxin 3 (PTX3) is an acute phase protein, involved in antibacterial resistance. Recent studies have shown PTX3 levels to be elevated in the presence of a bacterial infection and in a murine sepsis model. OBJECTIVE: We aim to investigate if sputum PTX3 can be used as a biomarker for bacterial infection in subjects with COPD. MATERIALS AND METHODS: Sputum samples from 142 COPD patients (102 men) with a mean (range) age of 69 years (45-85) and mean (SD) post-bronchodilator percentage predicted forced expiratory volume in 1 second (FEV1) of 50% (19) were analyzed for PTX3, using a commercial assay at stable state and during an exacerbation. Association with bacteria, from culture, quantitative real-time polymerase chain reaction (qPCR) and colony-forming units (CFU) was investigated. RESULTS: The geometric mean (95% CI) PTX3 level at stable state was 50.5 ng/mL (41.4-61.7). PTX3 levels correlated with absolute neutrophil count in sputum (r=0.37; P10(5) CFU/mL at stable state) with a receiver-operating characteristic (ROC) area under the curve (AUC) of 0.59 and 95% confidence interval (CI) 0.43-0.76 (P=0.21). During an exacerbation, there was a modest increase in PTX3 (fold difference 0.15, 95% of difference 0.02-0.29; P=0.02), and PTX3 fared better at identifying a bacteria-associated exacerbation (ROC AUC 0.65, 95% CI 0.52-0.78, P=0.03). CONCLUSION: PTX3 is associated with bacterial infection in patients with COPD, but its utility as a biomarker for identifying a bacteria-associated exacerbation warrants further studies

The Use of Exhaled Nitric Oxide to Guide Asthma Management: A Randomised Controlled Trial.

Rationale: Current asthma guidelines recommend adjusting antiinflammatory treatment on the basis of the results of lung function tests and symptom assessment, neither of which are closely associated with airway inflammation. Objectives: We tested the hypothesis that titrating corticosteroid dose using the concentration of exhaled nitric oxide in exhaled breath (FENO) results in fewer asthma exacerbations and more efficient use of corticosteroids, when compared with traditional management. Methods: One hundred eighteen participants with a primary care diagnosis of asthma were randomized to a single-blind trial of corticosteroid therapy based on either FENO measurements (n = 58) or British Thoracic Society guidelines (n = 60). Participants were assessed monthly for 4 months and then every 2 months for a further 8 months. The primary outcome was the number of severe asthma exacerbations. Analyses were by intention to treat. Measurements and Main Results: The estimated mean (SD) exacerbation frequency was 0.33 per patient per year (0.69) in the FENO group and 0.42 (0.79) in the control group (mean difference, –21%; 95% confidence interval [CI], –57 to 43%; p = 0.43). Overall the FENO group used 11% more inhaled corticosteroid (95% CI, –17 to 42%; p = 0.40), although the final daily dose of inhaled corticosteroid was lower in the FENO group (557 vs. 895 µg; mean difference, 338 µg; 95% CI, –640 to –37; p = 0.028). Conclusions: An asthma treatment strategy based on the measurement of exhaled nitric oxide did not result in a large reduction in asthma exacerbations or in the total amount of inhaled corticosteroid therapy used over 12 mo, when compared with current asthma guidelines