Project description:Background and objectiveSome children with asthma have low lung growth, putting them at increased risk for COPD later in life. However, it is currently not clear who will experience this adverse growth pattern. We therefore investigated the predictive role of blood eosinophils as a type 2 inflammation marker in lung growth, focusing on the presence and severity of asthma.MethodsWe investigated blood eosinophils and lung function growth (percentage of predicted values) using linear mixed models in children and adolescents from two longitudinal cohorts. One cohort was hospital-based and consisted of asthmatic children at their first outpatient clinic visit after referral by the general practitioner (n = 133, mean age 9.8), while the second was a general population-based birth cohort (PIAMA, asthma n = 52 and non-asthma n = 433, mean age 8.1). The hospital-based cohort had not been treated with inhaled corticosteroids (ICS) before referral.ResultsSubjects in the hospital-based asthma cohort had more severe asthma compared with the asthmatic subjects in the population-based cohort, defined by lower lung function levels and a higher prevalence of bronchial hyper-responsiveness. In the asthma cohort, higher blood eosinophil numbers were associated with less growth in FEV1 (estimated change in lung function per 1 unit increase in ln blood eosinophils (B): -0.66%/year (95% confidence interval (CI): -1.11 to -0.20, p < .01)) and FVC (B: -0.40%/year (95% CI: -0.75 to -0.05), p = .025)) during follow-up in adolescence (min 7, max 17 years). These associations were not observed in the general population-based birth cohort, regardless of asthma status during follow-up (age 8-16).Conclusions and clinical relevanceBlood eosinophil counts in children with asthma not treated with ICS at referral were predictive of lower growth in FEV1 and FVC during follow-up in adolescence. Our findings indicate that this association is dependent on the degree of asthma severity. Future studies should address whether anti-eosinophilic treatments preserve lung function growth in children with asthma.

Project description:Eosinophilia is a characteristic feature of allergic airway inflammation and remodeling. Chloride channel-3 (CLC3) in eosinophils has been associated with superoxide generation and respiratory burst. The CLC3 gene may produce multiple transcript variants through alternative splicing. However, the presence of CLC3 variants in human eosinophils is unknown. We examined the expression of CLC3 transcript variants in peripheral blood eosinophils of allergic asthmatics and healthy individuals. Potential of these obligatory dimers to form homo- or hetero-dimers was examined in HEK293 cells co-transfected with CLC3b-GFP and CLC3e-RFP. Eosinophils were isolated from peripheral blood by negative selection. Expression of CLC3 and CLC3 transcript variants was examined by qPCR, Western blot, and immunofluorescence. Confocal micrographs were analyzed with Image J software. Higher levels of novel transcript variants of CLC3 (CLC3b and CLC3e) were found in peripheral blood eosinophils of asthmatics compared to healthy non-atopic subjects. We also found higher CLC3 protein expression in the blood and nasal lavage eosinophils of asthmatics than healthy subjects. Both membranous and intracellular CLC3 expression were observed. Also, we found the presence of both homodimers and heterodimers of CLC3b-GFP and CLC3e-RFP in HEK293 cells. Higher and differential expression of novel CLC3 transcript variants in mild-to-moderate and moderate-to-severe asthmatic eosinophils suggest their critical role in allergic asthma. Membranous and intracellular (granular) expression of CLC3 in nasal lavage and peripheral blood eosinophils suggest their involvement in the activation and migration of eosinophils in allergic asthma. Moreover, homo- and hetero-dimerization of these transcript variants may change the channel properties to exhibit these states. Presence of CLC3 variants may serve as a biomarker in allergic asthma and additional knowledge of interaction between CLC3 transcript variants and their specific role in the activation and migration of eosinophils will allow to explore novel therapeutic approach in allergic asthma.

Project description:Background: Allergen inhalation challenge in mild asthmatics induces airflow obstruction, airway hyperresponsiveness and inflammation, providing a model for hypothesis-generating experiments to understand regulation of these responses. We have sought to evaluate the peripheral whole blood transcriptome, post-challenge compared to pre-challenge, and to determine the effect of globin mRNA reduction methodology. Methods: Asthmatic subjects (20-60 years of age, with stable, mild allergic asthma, n=17) underwent allergen inhalation challenges. All subjects had an early asthmatic response of ≥ 20% fall in FEV1; most had a late phase response of ≥ 15% fall in FEV1. Blood was collected immediately prior to, and two hours after allergen challenge. Transcriptome analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST arrays, with and without globin mRNA reduction (PAX-GR and PAX-NGR, respectively) in 4 subjects. Replication studies for expression of nine genes contributing to the top canonical pathway, Nrf2-mediated oxidative stress response pathway, were performed with 5 independent subjects with microarray data, also with the same 4 subjects had microarray results and 8 other independent subjects with QPCR. Data were analyzed using paired t-test and Partek® Genomics Suite™. Results: The number of differentially expressed probe sets from PAX-NGR samples was twice that of PAX-GR samples. Paired analysis of each subjects' pre- and post-sample additionally demonstrated twice the power to detect differentially expressed probe sets. The Nrf2-mediated oxidative stress response pathway was identified as the top canonical pathway in the PAX-NGR samples. ATP-binding cassette sub-family C member 1 (ABCC1) gene was significantly reduced two hours after allergen challenge in the 5 subjects’ microarray dataset and the 2 QPCR replication datasets with P < 0.05. Conclusions: Globin mRNA reduction does not provide benefits to detect differentially expressed genes during allergen inhalation challenge. Allergen inhalation challenge is associated with decreased peripheral blood cell transcript level of ABCC1 gene.

Project description:Background: Allergen inhalation challenge in mild asthmatics induces airflow obstruction, airway hyperresponsiveness and inflammation, providing a model for hypothesis-generating experiments to understand regulation of these responses. We have sought to evaluate the peripheral whole blood transcriptome, post-challenge compared to pre-challenge, and to determine the effect of globin mRNA reduction methodology. Methods: Asthmatic subjects (20-60 years of age, with stable, mild allergic asthma, n=17) underwent allergen inhalation challenges. All subjects had an early asthmatic response of ≥ 20% fall in FEV1; most had a late phase response of ≥ 15% fall in FEV1. Blood was collected immediately prior to, and two hours after allergen challenge. Transcriptome analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST arrays, with and without globin mRNA reduction (PAX-GR and PAX-NGR, respectively) in 4 subjects. Replication studies for expression of nine genes contributing to the top canonical pathway, Nrf2-mediated oxidative stress response pathway, were performed with 5 independent subjects with microarray data, also with the same 4 subjects had microarray results and 8 other independent subjects with QPCR. Data were analyzed using paired t-test and Partek® Genomics Suite™. Results: The number of differentially expressed probe sets from PAX-NGR samples was twice that of PAX-GR samples. Paired analysis of each subjects' pre- and post-sample additionally demonstrated twice the power to detect differentially expressed probe sets. The Nrf2-mediated oxidative stress response pathway was identified as the top canonical pathway in the PAX-NGR samples. ATP-binding cassette sub-family C member 1 (ABCC1) gene was significantly reduced two hours after allergen challenge in the 5 subjects’ microarray dataset and the 2 QPCR replication datasets with P < 0.05. Conclusions: Globin mRNA reduction does not provide benefits to detect differentially expressed genes during allergen inhalation challenge. Allergen inhalation challenge is associated with decreased peripheral blood cell transcript level of ABCC1 gene. 26 array were analyzed. We first identified differentially expreed genes between post- and pre-challenge samples from 4 asthmatic subjects by using globin reduction samples and non-globin reduction samples (16 arrays). Then we replicated our findings using 10 arrays from 5 asthmatic subjects challenged with allergen. Includes 4 normal blood samples.

Project description:BackgroundMicroRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level. While they have been implicated in various diseases, the profile changes in allergen inhalation challenge are not clarified in human. We aimed to evaluate changes in the microRNA profiles in the peripheral blood of asthmatic subjects undergoing allergen inhalation challenge.ResultsSeven mild asthmatic subjects participated in the allergen inhalation challenge. In addition, four healthy control subjects (HCs) were recruited. MicroRNA profiles in peripheral blood samples (pre-challenge and 2 hours post-challenge) were measured by the NanoString nCounter assay to determine changes in miRNA levels as these asthmatic subjects underwent an allergen inhalation challenge. One common miRNA, miR-192, was significantly expressed in both comparisons; HCs vs. pre-challenge and pre- vs. post-challenge, showing that miR-192 was significantly under-expressed in asthmatics compared to HCs and decreased in post-challenge at an FDR of 1%. Cell-specific statistical deconvolution attributed miR-192 expression in whole blood to PBMCs. MiR-192 was technically validated using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showing that the level in asthmatics (pre-challenge) was significantly lower than HCs and that post-challenge was significantly lower than pre-challenge. The normalized relative miR-192 expression quantified using RT-qPCR specific to PBMCs was also validated. Ontology enrichment and canonical pathway analyses for target genes suggested several functions and pathways involved in immune response and cell cycle.ConclusionsThe miRNA profile in peripheral blood was altered after allergen inhalation challenge. Change in miR-192 levels may be implicated in asthma mechanisms. These results suggest that allergen inhalation challenge is a suitable method to characterize peripheral miRNA profiles and potentially elucidate the mechanism of human asthma.

Dataset Information

Transcriptional Changes of Blood Eosinophils After Methacholine Inhalation Challenge in Asthmatics.

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Transcriptional Changes of Blood Eosinophils After Methacholine Inhalation Challenge in Asthmatics.

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