[Show abstract][Hide abstract] ABSTRACT: Eosinophil migration to the lung is primarily regulated by the eosinophil-selective family of eotaxin chemokines, which mobilize intracellular calcium (Ca(2+)) and orchestrate myriad changes in cell structure and function. Eosinophil function is also known to be flow-dependent, although the molecular cognate of this mechanical response has yet to be adequately characterized. Using confocal fluorescence microscopy, we determined the effects of fluid shear stress on intracellular calcium concentration ([Ca(2+)]i) in human peripheral blood eosinophils by perfusing cells in a parallel-plate flow chamber. Our results indicate that fluid perfusion evokes a calcium response that leads to cell flattening, increase in cell area, shape change, and non-directional migration. None of these changes are seen in the absence of a flow stimulus, and all are blocked by chelation of intracellular Ca(2+) using BAPTA. These changes are enhanced by stimulating the cells with eotaxin-1. The perfusion-induced calcium response (PICR) could be blocked by pre-treating cells with selective (CDP-323) and non-selective (RGD tripeptides) integrin receptor antagonists, suggesting that α4β7/α4β1 integrins mediate this response. Overall, our study provides the first pharmacological description of a molecular mechanosensor that may collaborate with the eotaxin-1 signaling program in order to control human eosinophil activation.
Frontiers in Immunology 11/2015; 6:525. DOI:10.3389/fimmu.2015.00525
[Show abstract][Hide abstract] ABSTRACT: OX40-OX40L interactions and TSLP are important in the induction and maintenance of Th2 responses in allergic disease whereas T regulatory cells (Treg) have been shown to suppress pro-inflammatory Th2 responses. Both OX40L and TSLP have been implicated in the negative regulation of Treg. The effect of anti-asthma therapies on Treg is not well known. Our aim then was to assess the effects of two monoclonal antibody therapies (anti-OX40L and anti-TSLP) on Treg frequency using a human model of allergic asthma. We hypothesized that the anti-inflammatory effects of these therapies would result in an increase in circulating Treg (CD4(+) CD25(+) CD127(low) Foxp3(+) cells) frequency. We measured Treg using flow cytometry and our results showed that neither allergen challenge nor monoclonal antibody therapy altered circulating Treg frequency. These data highlight the need for assessment of airway Treg and for a more complete understanding of Treg biology so as to develop pharmacologics/biologics that modulate Treg for asthma therapy. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Background:
In patients with severe eosinophilic asthma, local maturation rather than systemic recruitment of mature cells might contribute to persistent airway eosinophilia. Group 2 innate lymphoid cells (ILC2s) are a major source of type 2 cytokines (IL-5 and IL-13) and can facilitate eosinophilic inflammatory responses in mouse models of asthma in the absence of CD4(+) lymphocytes. This study investigated the potential role of ILC2s in driving chronic airway eosinophilia in patients with severe asthma, despite regular high-dose oral corticosteroid therapy.
In a cross-sectional study we enumerated blood and sputum ILC2s (lin(-)CD45(+)127(+)ST2(+)) and levels of intracellular IL-5 and IL-13 in patients with severe asthma (n = 25), patients with steroid-naive mild atopic asthma (n = 19), and nonatopic control subjects (n = 5). Results were compared with numbers of CD4(+) lymphocytes, eosinophil lineage-committed progenitors (eosinophilopoietic progenitor cells [EoPs]), and mature eosinophils.
Significantly greater numbers of total and type 2 cytokine-producing ILC2s were detected in blood and sputum of patients with severe asthma compared to mild asthmatics. In contrast, intracellular cytokine expression by CD4 cells and EoPs within the airways did not differ between the asthmatic groups. In patients with severe asthma, although sputum CD4(+) cells were more abundant than ILC2s and EoPs, proportionally, ILC2s were the predominant source of type 2 cytokines. In addition, there were significantly greater numbers of sputum IL-5(+)IL-13(+) ILC2s in patients with severe asthma whose airway eosinophilia was greater than 3%, despite normal blood eosinophil numbers (<300/μL).
Our findings suggest that ILC2s can promote the persistence of airway eosinophilia in patients with severe asthma through uncontrolled localized production of the type 2 cytokines IL-5 and IL-13, despite high-dose oral corticosteroid therapy.
The Journal of allergy and clinical immunology 07/2015; DOI:10.1016/j.jaci.2015.05.037 · 11.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the safety, tolerability and clinical activity of ASM-024, a new cholinergic compound with dual nicotinic and muscarinic activity, in mild allergic asthma.
The present study involved 24 stable, mild allergic asthmatic subjects. In a cross-over design, ASM-024 (50 mg or 200 mg) or placebo were administered once daily by nebulization over three periods of nine consecutive days separated by a three-week washout. The effect of each treatment on the forced expiratory volume in 1 s (FEV1), provocative concentration of methacholine causing a 20% decline in FEV1 (PC20), early and late asthmatic responses, and allergen-induced inflammation were measured.
Seventeen subjects completed the study. During treatment with ASM-024 at 50 mg or 200 mg, the PC20 value increased respectively from a mean (± SD) 2.56±3.86 mg⁄mL to 4.11 mg⁄mL (P=0.007), and from 3.12±4.37 mg⁄mL to 5.23 mg⁄mL (P=0.005) (no change with placebo). On day 7 (day preceding allergen challenge), postdosing FEV1 increased by 2.0% with 50 mg (P=0.005) and 1.9% with 200 mg (P=0.008) (placebo -1.1%). ASM-24 had no inhibitory effect on early and late asthmatic responses, nor on sputum eosinophil or neutrophil levels. ASM-024 induced no serious adverse events, but caused cough in 22% and 48% of the subjects with 50 mg and 200 mg, respectively, compared with 10% who were on placebo.
ASM-024 did not inhibit allergen-induced asthmatic response and related airway inflammation, but reduced methacholine airway responsiveness and slightly improved lung function. The mechanism by which ASM-024 improves these outcomes requires further study.
Canadian respiratory journal: journal of the Canadian Thoracic Society 07/2015; 22(4):230-4. · 1.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The allergen bronchoprovocation test (ABP) is a validated model to study asthma pathophysiology and response to treatments. The inhibitory effect of agents on the allergen-induced late asthmatic response (LAR) is a predictor of their efficacy in asthma treatment. However, it is difficult to predict the magnitude of a LAR, which may vary according to immune responsiveness and the type of allergen used for ABP.
To determine the relationship between the magnitudes of EAR and LAR in mild asthmatic subjects according to the type of allergen inhaled, and its determinants.
This is a retrospective analysis of a large database of ABPs, all performed with a common standardized methodology. Patients were either challenged with house-dust mites (HDM), animals, or pollens. EAR was defined as a ≥20% fall in forced expiratory volume in one second (FEV1 ) <3h following ABP and LAR as a ≥15% fall in FEV1 between 3h and 7h post ABP. The ratio of EAR% fall in FEV1 /LAR % fall in FEV1 was compared between the groups of subjects according to the allergen used for ABP.
Data from 290 subjects were analyzed: 87 had an isolated EAR and 203, a dual response (EAR+LAR). Dual responders had a significantly lower baseline PC20 , a more marked fall in FEV1 at EAR, and a trend towards higher baseline sputum eosinophil percentages. The ratio of EAR over LAR was significantly lower in HDM compared with pollen ABP, indicating a larger LAR for a similar EAR. No correlations were observed between the ratio of EAR over LAR and the various parameters recorded in the different groups analyzed.
Different mechanisms may be involved in modulating the magnitude of the LAR, according to the type of allergen. HDM seems to induce a stronger LAR than pollens, animal allergens being intermediary in this regard. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The ATS guidelines for methacholine testing for the diagnosis of asthma recommends the two minute tidal breathing protocol with the Wright nebuliser, which produces more aerosol than required, generates a small particle size, and requires cleaning between tests.
The objective was to evaluate methacholine testing using a disposable, breath-actuated AeroEclipse™ II, which produces aerosol during inspiration and was developed for single-patient use.
Forty-six adult asthmatic subjects (19 male), aged 27.3 (SD 9.5) years, with FEV1 98.5 (SD 18.1) % of predicted participated in a randomised, crossover, observational study. Subjects were first screened using the Wright nebulizer, then assigned to 2 minutes of tidal breathing from the Wright or 20 seconds of tidal breathing from the AeroEclipse™ nebuliser on 2 separate days, in random order. Methacholine PC20 values were calculated by linear interpolation of log dose versus response curves, log-transformed, and compared using paired student t-test and Pearson correlation.
The thirty eight subjects demonstrating reproducible PC20 measurements of within 1.5 doubling concentrations were included in the comparison. The geometric mean methacholine PC20 measured with the AeroEclipse™ nebuliser was approximately 1 doubling concentration lower than the geometric mean methacholine PC20 of the Wright nebuliser (p<0.05). The Pearson correlation coefficient between the two nebulisers was 0.86 (P<0.05).
The PC20 measurements using the two nebulisers were highly correlated, however, the PC20 determined with the AeroEclipse™ nebuliser was significantly lower than those determined using the Wright nebuliser.
Annals of the American Thoracic Society 04/2015; DOI:10.1513/AnnalsATS.201412-571BC
[Show abstract][Hide abstract] ABSTRACT: Mast cell, basophil, and eosinophil lineages all derive from CD34(+) hemopoietic stem cells; however, mast cells are derived from a distinct, nonmyeloid progenitor, while eosinophils and basophils share a common myeloid progenitor. These progenitors likely evolved from an ancestral leukocyte population involved in innate immunity and currently play a central role in the pathology of allergic disease. Advances in isolation and analysis of mast cell and basophil/eosinophil progenitor populations have been critical to understanding lineage commitment, differentiation, function, and transcriptional regulation of these cells and have provided a way of monitoring the effect of novel investigational therapies on these cell populations in samples of blood, bone marrow, and airway secretions.
[Show abstract][Hide abstract] ABSTRACT: Rationale: Effective anti-inflammatory therapies are needed for the treatment of asthma, but preferably without the systemic adverse effects of glucocorticosteroids. Objectives: We evaluated the effect of an inhaled non-steroidal glucocorticoid receptor agonist, AZD5423, on allergen-induced responses. Methods : Twenty mild allergic asthmatic subjects were randomized to receive 7 days of treatment with nebulized AZD5423 (75 μg or 300 μg) once daily, budesonide 200 μg twice daily via Turbuhaler®, and placebo in a double-blind, four-period, cross-over design study (NCT01225549). Allergen challenge was performed on Day 6. Measurements: FEV1 was measured repeatedly for 7 hours post-allergen for early and late asthmatic responses. Sputum inflammatory cells were measured before and at 7h and 24h post-allergen, and methacholine airway responsiveness was measured before and 24h post-allergen. Main Results: AZD5423 significantly attenuated the fall in FEV1 during the late asthmatic response (both doses 8.7% fall) versus placebo (14% fall) (p<0.05) with no effect of budesonide (12.5% fall) versus placebo (p>0.05). There was no effect on the fall in FEV1 during early asthmatic response. AZD5423 300 μg and 75 μg significantly attenuated allergen-induced sputum eosinophilia by 63% and 61% at 7h, and by 46% and 34% at 24h post-allergen, respectively, versus placebo (all p<0.05). Budesonide did not reduce allergen-induced sputum eosinophilia versus placebo. AZD5423 at 300 μg significantly attenuated allergen-induced airway hyperresponsiveness at 24h post-allergen versus placebo (p<0.05). Both doses of AZD5423 were well tolerated. Conclusions: Seven days inhalation of the non-steroidal glucocorticoid receptor agonist AZD5423 effectively reduced allergen-induced responses in subjects with mild allergic asthma. Clinical trial registration available at www.clinicaltrials.gov, ID NCT01225549.
American Journal of Respiratory and Critical Care Medicine 12/2014; 191(2). DOI:10.1164/rccm.201404-0623OC · 13.00 Impact Factor