Nonasthmatic Subjects Research Articles

Mannose receptor modulates macrophage polarization and allergic inflammation through miR-511-3p

Mannose receptor (MRC1/CD206) has been suggested to mediate allergic sensitization and asthma to multiple glycoallergens, including cockroach allergens. We sought to determine the existence of a protective mechanism through which MRC1 limits allergic inflammation through its intronic miR-511-3p. We examined MRC1-mediated cockroach allergen uptake by lung macrophages and lung inflammation using C57BL/6 wild-type (WT) and Mrc1-/- mice. The role of miR-511-3p in macrophage polarization and cockroach allergen-induced lung inflammation in mice transfected withadeno-associated virus (AAV)-miR-511-3p (AAV-cytomegalovirus-miR-511-3p-enhanced green fluorescent protein) was analyzed. Gene profiling of macrophages with or without miR-511-3p overexpression was also performed. Mrc1-/- lung macrophages showed a significant reduction in cockroach allergen uptake compared with WT mice, and Mrc1-/- mice had an exacerbated lung inflammation with increased levels of cockroach allergen-specific IgE and TH2/TH17 cytokines in a cockroach allergen-induced mouse model compared with WT mice. Macrophages from Mrc1-/- mice showed significantly reduced levels of miR-511-3 and an M1 phenotype, whereas overexpression of miR-511-3p rendered macrophages to exhibit a M2 phenotype. Furthermore, mice transfected with AAV-miR-511-3p showed a significant reduction in cockroach allergen-induced inflammation. Profiling of macrophages with or without miR-511-3p overexpression identified 729 differentially expressed genes, wherein expression of prostaglandin D2 synthase (Ptgds) and its product PGD2 were significantly downregulated by miR-511-3p. Ptgds showed a robust binding to miR-511-3p, which might contribute to the protective effect of miR-511-3p. Plasma levels of miR-511-3p were significantly lower in human asthmatic patients compared with nonasthmatic subjects. These studies support a critical but previously unrecognized role of MRC1 and miR-511-3p in protection against allergen-induced lung inflammation.

Immunohistology and remodeling in fatal pediatric and adolescent asthma

BackgroundThickening of reticular basement membrane, increased airway smooth muscle mass and eosinophilic inflammation are found in adult fatal asthma. At the present study the histopathology of fatal paediatric and adolescent asthma is evaluated.MethodsPost-mortem lung autopsies from 12 fatal asthma cases and 8 non-asthmatic control subjects were examined. Thickness of reticular basement membrane (RBM) and percentage of airway smooth muscle (ASM%) mass area were measured and inflammatory cells were counted. Patient records were reviewed for clinical history.ResultsThe age range of the cases was from 0.9 to 19.5 years, eight were males and five had received inhaled corticosteroids. Thickened RBM was detected in majority of the cases without any correlation to treatment delay, age at onset of symptoms or diagnosis. In the large airways ASM was clearly increased in one third of the cases whereas the median ASM% did not differ from that in healthy controls (14.0% vs. 14.0%). In small airways no increase of ASM was found, instead mucous plugs were seen in fatal asthma. The number of eosinophils, plasmacytoid dendritic cells, macrophages, and B-cells were significantly increased in fatal asthma cases compared with controls and the two latter correlated with the length of the fatal exacerbation.ConclusionsThe findings highlight the strong presence of eosinophils and mucous plugs even in small airways in children and adolescents with fatal asthma. Thickened RBM was obvious in majority of the patients. Contrary to our hypothesis, increased ASM% was detected in only one third of the patients.

Diagnostic accuracy of fractional exhaled nitric oxide measurement in predicting cough-variant asthma and eosinophilic bronchitis in adults with chronic cough: A systematic review and meta-analysis

Individual studies have suggested the utility of fractional exhaled nitric oxide (Feno) measurement in detecting cough-variant asthma (CVA) and eosinophilic bronchitis (EB) in patients with chronic cough. We sought to obtain summary estimates of diagnostic test accuracy of Feno measurement in predicting CVA, EB, or both in adults with chronic cough. Electronic databases were searched for studies published until January 2016, without language restriction. Cross-sectional studies that reported the diagnostic accuracy of Feno measurement for detecting CVA or EB were included. Risk of bias was assessed with Quality Assessment of Diagnostic Accuracy Studies 2. Random effects meta-analyses were performed to obtain summary estimates of the diagnostic accuracy of Feno measurement. A total of 15 studies involving 2187 adults with chronic cough were identified. Feno measurement had a moderate diagnostic accuracy in predicting CVA in patients with chronic cough, showing the summary area under the curve to be 0.87 (95% CI, 0.83-0.89). Specificity was higher and more consistent than sensitivity (0.85 [95% CI, 0.81-0.88] and 0.72 [95% CI, 0.61-0.81], respectively). However, in the nonasthmatic population with chronic cough, the diagnostic accuracy to predict EB was found to be relatively lower (summary area under the curve, 0.81 [95% CI, 0.77-0.84]), and specificity was inconsistent. The present meta-analyses indicated the diagnostic potential of Feno measurement as a rule-in test for detecting CVA in adult patients with chronic cough. However, Feno measurement may not be useful to predict EB in nonasthmatic subjects with chronic cough. These findings warrant further studies to validate the roles of Feno measurement in clinical practice of patients with chronic cough.

Assessment of Nutritional Status of Patients Suffering from Asthma

Background and aim: Several studies have suggested that increasing prevalence of asthma could be associated with nutrients intake. These nutrients can play beneficial as well as detrimental role in asthma and may affect the nutritional status of asthma patients. This case-control study was undertaken to assess the nutritional status and differences in dietary intake between a population with and without asthma. Methods: A case-control study was carried out in clinically and spirometrically diagnosed asthma patients and (n=143, average age 29.35 ± 8.64 years) and age, sex and socioeconomic status matched healthy volunteers (n=143, average age 29.54 ± 8.61 years) to assess and evaluate the nutritional status of asthmatics. Anthropometric measurements(BMI, MUAC, and TSF), biochemical investigations (Hb, total and differential leucocyte counts and serum calcium and protein and serum IgE), pulmonary function test, nutrient intake (3 day diary method and food frequency questionnaire) and subjective global assessment were used asstandard nutritional assessment tools. Results were considered statistically significant if the observed two-sided significance level (P-value) was not greater than 0.05. Results: Thepresentstudy resultsfoundthatthemajorityof casesweresignificantly overweight or obese on the basis of body mass index (p=0.01), mid upper arm circumference (p=0.01) and triceps skin fold thickness (p=0.01) than controls. Hb, serum protein and serum calcium, levels were significantly lower in asthma cases in comparison of non-asthmatic controls (p=0.01, 0.01, 0.01 respectively) and TLC, eosinophils, monocytes and serum Ig E levels were significantly higher in asthmatics in comparison to nonasthmatics (p=0.01, 0.01, 0.01 and 0.00 respectively). Daily intake of fat was significantly higher whereas protein intake was significantly lower in asthmatics than in non-asthmatic subjects (p=0.01 and p=0.01 respectively). Significant decrease in daily intakes of thiamine, riboflavin, vitamin C, calcium and iron was noticed in asthmatic than non-asthmatic subjects (p=0.01, p=0.02, p=0.01, p=0.01, p=0.01, p=0.00). Based on subjective global assessment rating scale, significant difference was observed between the groups (p=0.04) as 48.9% and 21.7% of the asthmatics and 23.6% and 47% of the nonasthmatics were moderately malnourished and well-nourished respectively. Conclusion: It can be concluded that overall nutritional status of asthma patients was poor based on the standard tools used. Most of them were obese, had unbalanced nutrient intake and were not well nourished. Thus the time may be close for consideration of dietary advice as per recommended and modified dietary allowances followed by intervention trials.

Does chronic physical activity level modify the airway inflammatory response to an acute bout of exercise in the postprandial period?

Recent studies have confirmed that a single high-fat meal (HFM) leads to increased airway inflammation. However, exercise is a natural anti-inflammatory and may modify postprandial airway inflammation. The postprandial airway inflammatory response is likely to be modified by chronic physical activity (PA) level. This study investigated whether chronic PA modifies the airway inflammatory response to an acute bout of exercise in the postprandial period in both insufficiently active and active subjects. Thirty-nine nonasthmatic subjects (20 active, 13 males/7 females) who exceeded PA guidelines (≥150 min moderate-vigorous PA/week) and 19 insufficiently active (6 males/13 females) underwent an incremental treadmill test to exhaustion to determine peak oxygen uptake. Subjects were then randomized to a condition (COND), either remaining sedentary (CON) or exercising (EX) post-HFM. Exercise was performed at the heart rate corresponding to 60% peak oxygen uptake on a treadmill for 1 h post-HFM (63% fat, 10 kcal/kg body weight). Blood lipids and exhaled nitric oxide (eNO: marker of airway inflammation) were measured at baseline and 2 h and 4 h post-HFM. Sputum differential cell counts were performed at baseline and 4 h post-HFM. The mean eNO response for all groups increased at 2 h post-HFM (∼6%) and returned to baseline by 4 h (p = 0.03). There was a time × COND interaction (p = 0.04), where EX had a greater eNO response at 4 h compared with CON. Sputum neutrophils increased at 4 h post-HFM (p < 0.05). These findings suggest that airway inflammation occurs after an HFM when exercise is performed in the postprandial period, regardless of habitual activity level.

Coexistence of obesity and asthma determines a distinct respiratory metabolic phenotype

Epidemiologic and clinical evidence supports the existence of an obesity-related asthma phenotype. No distinct pathophysiologic elements or specific biomarkers have been identified thus far, but increased oxidative stress has been reported. We aimed at verifying whether metabolomics of exhaled breath condensate from obese asthmatic (OA) patients, lean asthmatic (LA) patients, and obese nonasthmatic (ONA) subjects could recognize specific and statistically validated biomarkers for a separate "asthma-obesity" respiratory metabolic phenotype, here defined as "metabotype." Twenty-five OA patients, 30 ONA subjects, and 30 mild-to-moderate LA age-matched patients participated in a cross-sectional study. Nuclear magnetic resonance (NMR) profiles were analyzed by using partial least-squares discriminant analysis, and the results were validated with an independent patient set. From NMR profiles, we obtained strong regression models that distinguished OA patients from ONA subjects (quality parameters: goodness-of-fit parameter [R2]=0.81 and goodness-of-prediction parameter [Q2]=0.79), as well as OA patients from LA patients (R2=0.91 and Q2=0.89). The all-classes comparison (R2=0.86 and Q2=0.83) indicated that OA patients possess a respiratory metabolic profile fully divergent from those obtained in the other patient groups. We also identified specific biomarkers for between-class separation, which are independent from clinical bias. They are involved in the methane, pyruvate, and glyoxylate and dicarboxylate metabolic pathways. NMR-based metabolomics indicates that OA patients are characterized by a respiratory metabolic fingerprint fully different from that of patients independently affected by asthma or obesity. Such a phenotypic difference strongly suggests unique pathophysiologic pathways involved in the pathogenesis of asthma in adult obese subjects. Furthermore, the OA metabotype could define a strategy for patient stratification based on unbiased biomarkers, with important diagnostic and therapeutic implications.

Effect of aging on sputum inflammation and asthma control

Aged asthmatic patients experience increased morbidity and mortality. Knowledge of the aging effect on airway inflammation and asthma control is limited. We sought to compare airway inflammation and its relationship to asthma control in aged versus younger patients and determine whether differences are asthma specific or caused by "inflamm-aging." We performed a prospective study of aged (>60years) and younger (21-40years) inner-city patients with asthma. After a run-in period to control for inhaled corticosteroid use, induced sputum was collected. Age-matched nonasthmatic control subjects were included to measure age-related inflammatory changes. Aged (mean age, 67.9±5.1years; n=35) compared with younger (mean age, 30.8±5.9years; n=37) asthmatic patients had significantly worse asthma control and lower FEV1. Aged asthmatic patients had higher sputum neutrophil (30.5×104/mL and 23.1%) and eosinophil (7.0×104/mL and 3.8%) numbers and percentages compared with younger patients (neutrophils, 13.0×104/mL [P<.01] and 6.9% [P<.01]; eosinophils, 2.0×104/mL [P<.01] and 1.2% [P<.01]). Aged asthmatic patients had higher sputum IL-6 (P<.01) and IL-8 (P=.01) levels. No significant inflammatory differences between aged and younger control subjects were observed. In aged asthmatic patients increased sputum IL-6 and macrophage inflammatory protein 3α/CCL20 levels were significantly associated with decreased asthma control and increased sputum neutrophil numbers and IL-1β, IL-6, and macrophage inflammatory protein 3α/CCL20 levels were associated with hospitalization. The inflammatory patterns of aged versus younger asthmatic patients are associated with increased sputum neutrophil and eosinophil values and cytokine levels related to neutrophil recruitment. Differences in airway inflammation can contribute to diminished asthma control in the aged. Further understanding of asthma pathophysiology in aged patients is needed to improve management of this vulnerable population.

Excess medical costs in patients with asthma and the role of comorbidity

Asthmatic patients frequently have comorbidities, but the role of comorbidities in the economic burden of asthma is unclear. We examined the excess direct medical costs, including asthma- and comorbidity-related costs, in patients with asthma.We created a propensity score-matched cohort of patients newly diagnosed with asthma and non-asthmatic comparison subjects, both aged 5-55 years, from health administrative data (1997-2012) in British Columbia, Canada. Health services use records were categorised into 16 major disease categories based on International Classification of Diseases codes. Excess costs (in 2013 Canadian dollars ($)) were estimated as the adjusted difference in direct medical costs between the two groups.Average overall excess costs were estimated at $1058/person-year (95% CI 1006-1110), of which $134 (95% CI 132-136) was attributable to asthma and $689 (95% CI 649-730) to major comorbidity classes. Psychiatric disorders were the largest component of excess comorbidity costs, followed by digestive disorders, diseases of the nervous system, and respiratory diseases other than asthma. Comorbidity-attributable excess costs greatly increased with age but did not increase over the time course of asthma.These findings suggest that both asthma and comorbidity-related outcomes should be considered in formulating evidence-based policies and guidelines for asthma management.

ASCIA‐P49: SAFETY OF 300IR SUBLINGUAL TABLET OF HOUSE DUST MITE ALLERGEN EXTRACTS IN ADOLESCENTS WITH HOUSE DUST MITE‐ASSOCIATED ALLERGIC RHINITIS

Safety of house dust mite (HDM) sublingual tablets for the treatment of HDM-associated allergic rhinitis (AR) has been assessed across the clinical program. Here we present pooled safety data in adolescents treated with the 300IR dose. Adolescents (12–17 years) with medically confirmed HDM-associated AR were randomized in two Phase II/III double-blind studies conducted in Europe and Japan to receive a 300IR HDM tablet or placebo once daily for one year. Adverse events (AEs) were monitored throughout the studies and analyzed descriptively. 345 adolescents (300IR=173, Placebo = 172) comprised the Safety Set including 150 (43%) subjects with intermittent asthma at enrollment. The most commonly reported AEs during active treatment in the adolescents were mild or moderate application-site reactions such as throat irritation (13.9%), oral pruritus (9.8%) or mouth edema (8.7%). They occurred during the first day of treatment in nearly 20% of subjects and within the first month in about 50% of them. None of the serious AEs reported in 11 subjects (300IR=5, Placebo = 6) were considered drug-related. 18 subjects [300IR=12 (6.9%) and Placebo = 6 (3.5%)] withdrew due to an AE, mainly as a result of application-site reactions (e.g., upper abdominal pain, tongue edema). There were no reports of anaphylaxis and no use of epinephrine. Percentages of adolescents reporting at least one AE during active treatment or those reporting adverse reactions (ADRs) were similar in subjects with or without asthma [with asthma: 77% (AEs) and 50% (ADRs); without asthma: 78% (AEs) and 56% (ADRs)]. In both asthmatic and non-asthmatic subjects, the most frequent adverse reactions were those reported at the application site and were of similar incidences. A favorable safety profile of 300IR HDM sublingual tablets was demonstrated in adolescents with HDM-induced allergic rhinitis associated or not with intermittent asthma.

House Dust Mite Allergens: New Discoveries and Relevance to the Allergic Patient.

Recent findings on house dust allergens and their contribution to knowledge that will significantly impact on current and future allergy treatments are appraised. Quantitation of IgE binding to a spectrum of allergen components in several independent studies in varying locations has largely affirmed the main components as the groups 1 and 2 and possibly 23 allergens with mid-tier contributions from the groups 4, 5, 7, and 21. Prevalent binding to Der p 23 has been recapitulated sometimes with low titers. The IgE of non-asthmatic atopic subjects binds at lower titer and to fewer components than that of asthmatics, and their IgG binding relative to IgE is higher especially for children hospitalized for exacerbation. The higher IgG ratios were associated with increased IL-10 a cytokine more readily induced from T cells of allergic subjects. Peptides representing the groups 1 and 2 allergens can be used to stimulate ex vivo T cells showing responses correlating with IgE binding and providing a valuable tool for ascertaining the contribution of IgE and T cells to disease. Also, the induction of Th2 and follicular helper T cells are shown to make different contributions in mice. Cross-reactivity of IgE binding assays with high-titer cross-reactive antibodies induced by scabies is a problem in the many areas of the world where scabies is highly prevalent and endemic and from recent increases in immigration. In the last few years, allergen research has produced results that warrant rapid translation into diagnostic tools and the formulation of allergen components for immunotherapy.

Eosinophils enhance WNT-5a and TGF-β1 genes expression in airway smooth muscle cells and promote their proliferation by increased extracellular matrix proteins production in asthma

BackgroundRecent studies have suggested that eosinophils may have a direct effect on airway smooth muscle cells (ASMC), causing their proliferation in patients with asthma, but the precise mechanism of the interaction between these cells remains unknown. We propose that changes in Wnt signaling activity and extracellular matrix (ECM) production may help explain these findings. Therefore, the aim of this study was to investigate the effect of eosinophils from asthmatic and non-asthmatic subjects on Wnt-5a, transforming growth factor β1 (TGF-β1), and ECM protein (fibronectin and collagen) gene expression and ASMC proliferation.MethodsA total of 18 subjects were involved in the study: 8 steroid-free asthma patients and 10 healthy subjects. Peripheral blood eosinophils were isolated using centrifugation and magnetic separation. An individual co-culture of eosinophils with human ASMC was prepared for each study subject. Adhesion of eosinophils to ASMC (evaluated by assaying eosinophil peroxidase activity) was determined following various incubation periods (30, 45, 60, 120, and 240 min). The expression of Wnt-5a, TGF-β1, and ECM protein genes in ASMC was measured using quantitative real-time polymerase chain reaction (PCR) after 24 h of co-culture. Proliferation of ASMC was measured using the Alamar blue method after 48 h and 72 h of co-culture with eosinophils.ResultsEosinophils from asthmatic subjects demonstrated increased adhesion to ASMC compared with eosinophils from healthy subjects (p < 0.05) in vitro. The expression of Wnt-5a, TGF-β1, collagen, and fibronectin genes in ASMC was significantly higher after 24 h of co-culture with eosinophils from asthmatic subjects, while co-culture of ASMC with eosinophils from healthy subjects increased only TGF-β1 and fibronectin gene expression. ASMC proliferation was augmented after co-culture with eosinophils from asthma patients compared with co-culture with eosinophils from healthy subjects (p < 0.05).ConclusionsEosinophils enhance Wnt-5a, TGF-β1, fibronectin, and collagen gene expression in ASMC and promote proliferation of these cells in asthma.Trial registrationClinicalTrials.gov Identifier: NCT02648074.

Reduced transforming growth factor β1 (TGF-β1) in the repair of airway epithelial cells of children with asthma.

Evidence into the role of TGF-β1 in airway epithelial repair in asthma is still controversial. This study tested the hypothesis that the reduced TGF-β1 levels previously observed in paediatric asthmatic airway epithelial cells directly contribute to the dysregulated repair seen in these cells. Primary airway epithelial cells (pAEC) from children with asthma (n = 16) and non-asthmatic subjects (n = 20) were isolated, and subcultured for investigation of TGF-β1 gene and protein via quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression of other associated genes such as integrins αvβ6, αvβ8 and MT1-MMP were also tested. Small interfering RNA (siRNA) was employed to assess the role of TGF-β1 during wound repair. TGF-β1 gene and protein expression were significantly downregulated in asthmatic pAEC over the course of repair, compared with cells from non-asthmatic children. Messenger RNA (mRNA) expression of TGF-β1 was also directly implicated in non-asthmatic and asthmatic pAEC proliferation over their quiescent counterparts. Small interfering RNA-mediated knockdown of TGF-β1 compromised repair in non-asthmatic pAEC and exacerbated the dysregulated repair seen in asthmatic pAEC. Expression of major TGF-β1 activators of epithelial cells, integrin αvβ6 and αvβ8 was also measured and there was no difference in αvβ6 gene expression between the two cohorts. Although integrin αvβ8 gene expression was significantly higher in asthmatic pAEC, the expression of MT1-MMP (MMP14) which facilitates the αvβ8 mediated TGF-β1 activation was significantly downregulated. Our data has highlighted the importance of TGF-β1 in pAEC wound repair in vitro. The significantly lower levels seen in asthmatic pAEC subsequently contributes to the dysregulated repair observed in these cells.

IgE anti-Haemophilus influenzae type b (Hib) antibodies detected in serum of Hib-vaccinated asthmatic and non-asthmatic pediatric patients.

Haemophilus influenzae type b (Hib) bacterium causes severe illness in infants and children, but has largely been eliminated by introducing a universal Hib conjugate vaccine. While effects of certain vaccinations on atopic disease have been studied, little is known about the relationship between Hib vaccination and diseases of altered immunoglobulin E (IgE) regulation (asthma or atopy). As such, it is necessary to provide more evidence concerning Hib vaccination as a possible risk factor for atopic disease. Total serum IgE and IgE-and IgG-anti-Hib antibody responses were studied in Hib vaccinated asthmatic (N.=14) and non-asthmatic children (N=26) (VaccZyme™ Human Anti Hib Enzyme Immunoassay Kit). Data are reported as mean optical density (OD) values. We found that: 1) total serum IgE levels were higher in asthmatic compared with non-asthmatic subjects (389±125 vs. 125±129, P<0.001); 2) IgE and IgG anti-Hib antibody responses were similar in both asthmatic and non-asthmatic subjects (0.722±0.279 and 0.681±0.280, respectively; P=0.65; 0.450±0.505 and 0.573±0.779, respectively; P=0.580). The universal Hib vaccine antigen did not result in either increased IgE, or IgG anti-Hib antibody responses in asthmatic or non-asthmatics subjects. Thus, in this cohort, no association between Hib vaccination and asthma status was identified.

No effect of elevated operating lung volumes on airway function during variable workrate exercise in asthmatic humans.

In asthmatic adults, airway caliber fluctuates during variable intensity exercise such that bronchodilation (BD) occurs with increased workrate whereas bronchoconstriction (BC) occurs with decreased workrate. We hypothesized that increased lung mechanical stretch would prevent BC during such variable workrate exercise. Ten asthmatic and ten nonasthmatic subjects completed two exercise trials on a cycle ergometer. Both trials included a 28-min exercise bout consisting of alternating four min periods at workloads equal to 40 % (Low) and 70% (High) peak power output. During one trial, subjects breathed spontaneously throughout exercise (SVT), such that tidal volume (VT) and end-inspiratory lung volume (EILV) were increased by 0.5 and 0.6 liters during the high compared with the low workload in nonasthmatic and asthmatic subjects, respectively. During the second trial (MVT), VT and EILV were maintained constant when transitioning from the high to the low workload. Forced exhalations from total lung capacity were performed during each exercise workload. In asthmatic subjects, forced expiratory volume 1.0 s (FEV1.0) increased and decreased with the increases and decreases in workrate during both SVT (Low, 3.3 ± 0.3 liters; High, 3.6 ± 0.2 liters; P < 0.05) and MVT (Low, 3.3 ± 0.3 liters; High, 3.5 ± 0.2 liters; P < 0.05). Thus increased lung stretch during MVT did not prevent decreases in airway caliber when workload was reduced. We conclude that neural factors controlling airway smooth muscle (ASM) contractile activity during whole body exercise are more robust determinants of airway caliber than the ability of lung stretch to alter ASM actin-myosin binding and contraction.

CD151, a laminin receptor showing increased expression in asthmatic patients, contributes to airway hyperresponsiveness through calcium signaling

Airway smooth muscle (ASM) contraction underpins airway constriction; however, underlying mechanisms for airway hyperresponsiveness (AHR) remain incompletely defined. CD151, a 4-transmembrane glycoprotein that associates with laminin-binding integrins, is highly expressed in the human lung. The role of CD151 in ASM function and its relationship to asthma have yet to be elucidated. We sought to ascertain whether CD151 expression is clinically relevant to asthma and whether CD151 expression affects AHR. Using immunohistochemical analysis, we determined the expression of CD151 in human bronchial biopsy specimens from patients with varying asthma severities and studied the mechanism of action of CD151 in the regulation of ASM contraction and bronchial caliber invitro, exvivo, and invivo. The number of CD151+ ASM cells is significantly greater in patients with moderate asthma compared with those in healthy nonasthmatic subjects. From loss- and gain-of-function studies, we reveal that CD151 is required for and enhances G protein-coupled receptor (GPCR)-induced peak intracellular calcium release, the primary determinant of excitation-contraction coupling. We show that the localization of CD151 can also be perinuclear/cytoplasmic and offer an explanation for a novel functional role for CD151 in supporting protein kinase C (PKC) translocation to the cell membrane in GPCR-mediated ASM contraction at this site. Importantly, CD151-/- mice are refractory to airway hyperreactivity in response to allergen challenge. We identify a role for CD151 in human ASM contraction. We implicate CD151 as a determinant of AHR in vivo, likely through regulation of GPCR-induced calcium and PKC signaling. These observations have significant implications in understanding the mechanism for AHR and the efficacy of new and emerging therapeutics.

Circulating microRNAs as biomarkers in patients with allergic rhinitis and asthma

MicroRNAs (miRNAs) are emerging as important regulatory molecules that might be involved in the pathogenesis of various diseases. Circulating miRNAs might be noninvasive biomarkers to diagnose and characterize asthma and allergic rhinitis (AR). We sought to determine whether miRNAs are differentially expressed in the blood of asthmatic patients compared with those in the blood of nonasthmatic patients with AR and nonallergic nonasthmatic subjects. Furthermore, we sought to establish whether miRNAs could be used to characterize or subtype asthmatic patients. Expression of plasma miRNAs was measured by using real-time quantitative PCR in 35 asthmatic patients, 25 nonasthmatic patients with AR, and 19 nonallergic nonasthmatic subjects. Differentially expressed miRNAs were identified by using Kruskal-Wallis 1-way ANOVA with Bonferroni P value adjustment to correct for multiple comparisons. Arandom forest classification algorithm combined with a leave-one-out cross-validation approach was implemented to assess the predictive capacities of the profiled miRNAs. We identified 30 miRNAs that were differentially expressed among healthy, allergic, and asthmatic subjects. These miRNAs fit into 5 different expression pattern groups. Among asthmatic patients, miRNA expression profiles identified 2 subtypes that differed by high or low peripheral eosinophil levels. Circulating miR-125b, miR-16, miR-299-5p, miR-126, miR-206, and miR-133b levels were most predictive of allergic and asthmatic status. Subsets of circulating miRNAs are uniquely expressed in patients with AR and asthmatic patients and have potential for useas noninvasive biomarkers to diagnose and characterize these diseases.

Pharmacokinetics of nebulized and oral procaterol in asthmatic and non-asthmatic subjects in relation to doping analysis.

The purpose of the present study was to investigate pharmacokinetics of procaterol in asthmatics and non-asthmatics after nebulized and oral administration in relation to doping. Ten asthmatic and ten non-asthmatic subjects underwent two pharmacokinetic trials. At first trial, 4 µg procaterol was administered as nebulization. At second trial, 100 µg procaterol was administered orally. Serum and urine samples were collected before and after administration of procaterol. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum and urine concentrations of procaterol were markedly higher after oral administration compared to nebulized administration. After oral administration, serum procaterol concentration-time area under the curve (AUC) was higher (P ≤ 0.05) for asthmatics than non-asthmatics. Likewise, urine concentrations were higher (P ≤ 0.01) for asthmatics than non-asthmatics 4 (47 ± 12 vs. 28 ± 9 ng/mL) and 8 h (39 ± 9 vs. 15 ± 5 ng/mL) after oral administration. Detection of serum procaterol was difficult after nebulized administration with 38 samples (27%) below limit of quantification (LOQ) and only trends were observed. No differences were observed between asthmatics and non-asthmatics in the urine concentrations of procaterol after nebulized administration. In summary, our data showed that asthmatics had higher urine concentrations of procaterol than non-asthmatics after oral administration of 100 µg, whereas no difference was observed between the groups after nebulized administration. For doping control purposes, our observations indicate that it is possible to differentiate therapeutic nebulized administration of procaterol from prohibited use of oral procaterol. Copyright © 2016 John Wiley & Sons, Ltd.

Patterns of changes and diagnostic values of FEF50%, FEF25%-75% and FEF50%/FEF25%-75% ratio in patients with varying control of bronchial asthma.

The pattern of change in FEF50%, FEF25%-75% and FEF50%/FEF25%-75% ratio depends on mechanics of forceful expiration, provided that non-homogenous lung emptying is faithfully recorded in the expiratory flow-volume loops. To assess the potential clinical value of FEF50%, FEF25%-75% and FEF50%/FEF25%-75% ratio as an indicator of bronchial asthma (BA) control. The study involved 75 patients with BA matched for age and gender with 45 non-asthmatic subjects. Based on asthma control test (ACT) and spirometry, asthmatic patients were subdivided into controlled and poorly controlled/uncontrolled. The relationship between FEF50% and FEF25%-75% as well as FEF50%/FEF25%-75% ratio and ACT score were assessed using linear regression. ROC curves were used to assess reliability of FEF25%-75% and FEF50% to diagnose BA in patients with different degree of asthma control. FEF50% correlated strongly with FEF25%-75% (r = 0.989, P < 0.001) and the relationship between the two indices is governed by the formula FEF50% = 1.132* FEF25%-75% - 0.003. There was no significant correlation (r = - 0.159, P = 0.083) between FEF50%/FEF25-75% ratio and ACT score. The diagnostic capability of FEF25%-75% for spirometric diagnosis of BA is only marginally better compared to FEF50% (area under ROC curves were 0.88 and 0.89 respectively, P < 0.001); however, diagnostic power of both spirometric indices deceased with poor BA control. FEF50%/FEF25%-75% has no clinical value as an indicator for BA control. Role of FEF25%-75% in evaluation of BA is marginally better than FEF50%; however, efficiency of both indices declined substantially as BA control worsened.

Doublesex and mab-3 related transcription factor 1 (DMRT1) is a sex-specific genetic determinant of childhood-onset asthma and is expressed in testis and macrophages

Asthma is a disease affecting more boys than girls in childhood and more women than men in adulthood. The mechanisms behind these sex-specific differences are not yet understood. We analyzed whether and how genetic factors contribute to sex-specific predisposition to childhood-onset asthma. Interactions between sex and polymorphisms on childhood asthma risk were evaluated in the Multicentre Asthma Genetics in Childhood Study (MAGICS)/Phase II International Study of Asthma and Allergies in Childhood (ISAAC II) population on a genome-wide level, and findings were validated in independent populations. Genetic fine mapping of sex-specific asthma association signals was performed, and putatively causal polymorphisms were characterized invitro by using electrophoretic mobility shift and luciferase activity assays. Gene and protein expression of the identified gene doublesex and mab-3 related transcription factor 1 (DMRT1) were measured in different human tissues by using quantitative real-time PCR and immunohistochemistry. Polymorphisms in the testis-associated gene DMRT1 displayed interactions with sex on asthma status in a population of primarily clinically defined asthmatic children and nonasthmatic control subjects (lowest P=5.21×10(-6)). Replication of this interaction was successful in 2 childhood populations clinically assessed for asthma but showed heterogeneous results in other population-based samples. Polymorphism rs3812523 located in the putative DMRT1 promoter was associated with allele-specific changes in transcription factor binding and promoter activity invitro. DMRT1 expression was observed not only in the testis but also in lung macrophages. DMRT1 might influence sex-specific patterns of childhood asthma, and its expression in testis tissue and lung macrophages suggests a potential involvement in hormone or immune cell regulation.

Large-scale profiling of signalling pathways reveals an asthma specific signature in bronchial smooth muscle cells.

BackgroundBronchial smooth muscle (BSM) cells from asthmatic patients maintain in vitro a distinct hyper-reactive (“primed”) phenotype, characterized by increased release of pro-inflammatory factors and mediators, as well as hyperplasia and/or hypertrophy. This “primed” phenotype helps to understand pathogenesis of asthma, as changes in BSM function are essential for manifestation of allergic and inflammatory responses and airway wall remodelling.ObjectiveTo identify signalling pathways in cultured primary BSMs of asthma patients and non-asthmatic subjects by genome wide profiling of differentially expressed mRNAs and activated intracellular signalling pathways (ISPs).MethodsTranscriptome profiling by cap-analysis-of-gene-expression (CAGE), which permits selection of preferentially capped mRNAs most likely to be translated into proteins, was performed in human BSM cells from asthmatic (n=8) and non-asthmatic (n=6) subjects and OncoFinder tool were then exploited for identification of ISP deregulations.ResultsCAGE revealed >600 RNAs differentially expressed in asthma vs control cells (p≤0.005), with asthma samples showing a high degree of similarity among them. Comprehensive ISP activation analysis revealed that among 269 pathways analysed, 145 (p<0.05) or 103 (p<0.01) are differentially active in asthma, with profiles that clearly characterize BSM cells of asthmatic individuals. Notably, we identified 7 clusters of coherently acting pathways functionally related to the disease, with ISPs down-regulated in asthma mostly targeting cell death-promoting pathways and up-regulated ones affecting cell growth and proliferation, inflammatory response, control of smooth muscle contraction and hypoxia-related signalization.ConclusionsThese first-time results can now be exploited toward development of novel therapeutic strategies targeting ISP signatures linked to asthma pathophysiology.