Paul Cadden

Non-invasive phenotyping using exhaled volatile organic compounds in asthma

Background Breath volatile organic compounds (VOCs) may be useful for asthma diagnosis and phenotyping, identifying patients who could benefit from personalised therapeutic strategies. The authors aimed to identify specific patterns of breath VOCs in patients with asthma and in clinically relevant disease phenotypes. Methods Breath samples were analysed by gas chromatography–mass spectrometry. The Asthma Control Questionnaire was completed, together with lung function and induced sputum cell counts. Breath data were reduced to principal components, and these principal components were used in multiple logistic regression to identify discriminatory models for diagnosis, sputum inflammatory cell profile and asthma control. Results The authors recruited 35 patients with asthma and 23 matched controls. A model derived from 15 VOCs classified patients with asthma with an accuracy of 86%, and positive and negative predictive values of 0.85 and 0.89, respectively. Models also classified patients with asthma based on the following phenotypes: sputum (obtained in 18 patients with asthma) eosinophilia ≥2% area under the receiver operating characteristics (AUROC) curve 0.98, neutrophilia ≥40% AUROC 0.90 and uncontrolled asthma (Asthma Control Questionnaire ≥1) AUROC 0.96. Conclusions Detection of characteristic breath VOC profiles could classify patients with asthma versus controls, and clinically relevant disease phenotypes based on sputum inflammatory profile and asthma control. Prospective validation of these models may lead to clinical application of non-invasive breath profiling in asthma.

Inhibition of the asthmatic allergen challenge response by the CRTH2 antagonist OC000459

CRTH2 (chemoattractant receptor expressed on T-helper (Th) type 2 cells) is a G-protein-coupled receptor expressed by Th2 lymphocytes and eosinophils that mediates prostaglandin (PG)D2-driven chemotaxis. We studied the efficacy of the oral CRTH2 antagonist OC000459 in steroid-naïve asthmatic patients. A randomised, double-blind, placebo-controlled, two-way crossover study of 16 days’ treatment with OC000459 (200 mg twice daily) on the late (LAR) and early (EAR) asthmatic responses to bronchial allergen challenge was conducted, with 16 subjects completing the study. There was a 25.4% (95% CI 5.1–45.6%) reduction in the LAR area under the curve (AUC) for change in forced expiratory volume in 1 s with OC000459 compared with placebo (p=0.018) but no effect on the EAR. Sputum eosinophil counts at 1 day post-allergen challenge were lower after OC000459 treatment (p=0.002). PGD2-induced blood eosinophil shape change ex vivo was assessed at day 7 (n=7). The AUC of eosinophil shift for OC000459 was lower than placebo; the mean difference was -33.6% (95% CI -66.8– -0.4%; p=0.048). OC000459 treatment inhibited LAR and post-allergen increase in sputum eosinophils. This CRTH2 antagonist appears to inhibit allergic inflammation in asthma.

Synergistic Effects of p38 Mitogen-Activated Protein Kinase Inhibition with a Corticosteroid in Alveolar Macrophages from Patients with Chronic Obstructive Pulmonary Disease

Corticosteroids partially suppress cytokine production by chronic obstructive pulmonary disease (COPD) alveolar macrophages. p38 mitogen-activated protein kinase (MAPK) inhibitors are a novel class of anti-inflammatory drug. We have studied the effects of combined treatment with a corticosteroid and a p38 MAPK inhibitor on cytokine production by COPD alveolar macrophages, with the aim of investigating dose-sparing and efficacy-enhancing effects. Alveolar macrophages from 10 patients with COPD, six smokers, and six nonsmokers were stimulated with lipopolysaccharide (LPS) after preincubation with five concentrations of dexamethasone alone, five concentrations of the p38 MAPK inhibitor 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB-796) alone, and all combinations of these concentrations. After 24 h, the supernatants were analyzed for interleukin (IL)-8, IL-6, tumor necrosis factor α (TNFα), granulocyte macrophage–colony-stimulating factor (GM-CSF), IL-1α, IL-1β, IL-1ra, IL-10, monocyte chemoattractant protein 3, macrophage-derived chemokine (MDC), and regulated on activation normal T cell expressed and secreted (RANTES). The effect of dexamethasone on p38 MAPK activation was analyzed by Western blotting. Dexamethasone and BIRB-796 both reduced LPS-induced cytokine production in a dose-dependent manner in all subject groups, with no differences between groups. Increasing the concentration of BIRB-796 in combination with dexamethasone produced progressively greater inhibition of cytokine production than dexamethasone alone. There were significant efficacy-enhancing benefits and synergistic dose-sparing effects (p < 0.05) for the combination treatment for IL-8, IL-6, TNFα, GM-CSF, IL-1ra, IL-10, MDC, and RANTES in one or more subject groups. Dexamethasone had no effect on LPS-induced p38 MAPK activation. We conclude that p38 MAPK activation in alveolar macrophages is corticosteroid-insensitive. Combining a p38 MAPK inhibitor with a corticosteroid synergistically enhances the anti-inflammatory effects on LPS-mediated cytokine production by alveolar macrophages from patients with COPD and controls.

T lymphocyte insensitivity to corticosteroids in chronic obstructive pulmonary disease

BackgroundThere are increased numbers of activated lymphocytes in the lungs of chronic obstructive pulmonary disease (COPD) patients. The clinical benefits of corticosteroids in COPD patients are limited. Our hypothesis is that lymphocytes play a role in this corticosteroid insensitivity.ObjectivesTo investigate the effects of the corticosteroid dexamethasone on lung lymphocyte cytokine production from patients with COPD compared to controls.MethodsCultured airway lymphocytes obtained by bronchoscopy from healthy non-smokers (HNS), smokers (S) and COPD patients were stimulated with phytohaemagglutinin (PHA) & phorbol myristate acetate (PMA), +/- dexamethasone. Supernatants were assayed for interleukin (IL)-2 and interferon (IFN)γ. Immunofluoresence was used to analyse changes in CD8 glucocorticoid receptor (GRα and GRβ) expression.ResultsThe inhibition of PHA/PMA stimulated IFNγ production by dexamethasone was reduced in COPD patients compared to HNS (p < 0.05 at concentrations from 0.1-1 μM). There was also a significant reduction (p < 0.05) in the mean inhibitory effect at 1 μM in COPD patients (54.1%) compared to smokers (72.1%), and in smokers compared to HNS (85.5%). There was a numerically reduced effect of dexamethasone on IL-2 production that did not reach statistical significance. There was no difference in GRα and GRβ expression in follicular CD8 cells between COPD patients (50.9% and 30.4% respectively) and smokers (52.9% and 29.7% respectively).ConclusionsIFNγ production from COPD airway lymphocytes is corticosteroid insensitive. This phenomenon may be important in the poor clinical response often observed with corticosteroids.

Repeatability of induced sputum measurements in moderate to severe asthma.

BACKGROUND Novel therapies are being developed for patients with moderate to severe asthma. These patients may have neutrophilic airway inflammation. Induced sputum is commonly used as an endpoint in clinical trials of asthma therapies. We have performed repeated induced sputum sampling in moderate to severe asthma patients to understand the variability of cell counts, including neutrophils, and performed power calculations for studies in this group of patients. METHODS Nineteen patients with moderate to severe asthma with evidence of airflow obstruction (FEV1 ≤ 80% predicted) and suboptimal control (ACQ ≥ 1) performed repeated induced sputum separated by 1 month. RESULTS The Ri of neutrophil percentage and absolute eosinophil count demonstrated good (0.61) and moderate (0.56) repeatability respectively, but there was a poor level of agreement for eosinophil percentage and absolute neutrophil counts. The within subject SD for sputum neutrophil percentage was 15.8. In cross over studies, sample sizes of n = 14 and n = 54 are required to detect changes in neutrophil percentages by 20 and 10 % respectively at 90% power. CONCLUSIONS Sputum neutrophil percentage has good reproducibility in patients with moderate to severe asthma.

Leukotriene B4 levels in sputum from asthma patients

Poor asthma control is associated with increased airway neutrophils. Leukotriene B4 (LTB4) is a potent neutrophil chemoattractant. We examined the levels of LTB4 levels in the sputum of asthma patients and the relationship with disease severity. 47 asthma patients (categorised according to Global Initiative for Asthma treatment stage) and 12 healthy controls provided sputum samples that were processed first with PBS to obtain supernatants and secondly with dithiothreitol (DTT) to obtain supernatants. LTB4 levels were determined by ELISA. LTB4 levels were significantly higher in step 1 (steroid naïve) and step 3 (inhaled corticosteroid (ICS) plus long acting β-agonist) patients than step 2 patients (ICS alone) (p=0.02 and p=0.01, respectively). There was very good correlation when comparing PBS processed to DTT processed supernatants. High LTB4 levels were found in the sputum of asthmatics at step 3 despite ICS use. The levels of LTB4 are increased in the sputum of subgroups of asthma patients http://ow.ly/Xu6I303jVb5