Moderate-High Blood Eosinophilia Is Associated with Increased Hospitalization and Other Asthma Comorbidities.

(1) Background: Eosinophilia has traditionally been linked to eosinophilic asthma, for which it is the gold-standard prognostic biomarker. However, the association between eosinophilia and the presence of other diseases and comorbidities is yet unclear. (2) Methods: For this retrospective study, we reviewed the electronic medical records of 49,909 subjects with blood eosinophilia to gather data on the presence of asthma, COPD, sleep apnea, tuberculosis, dyslipidemia, hypertension, and other cardiovascular diseases and severe CRSwNP among these subjects. Demographic features including age, sex, and smoking habits were collected, as well as the number of hospitalizations and emergency department visits. T-tests, ANOVA, Fisher test, and logistic regression models were used. (3) Results: For all age groups studied, eosinophilia was significantly more prevalent among asthmatic subjects than nonasthmatics, especially in patients also presenting CRSwNP, hypertension, and dyslipidemia. The likelihood of developing asthma, COPD, and CRSwNP, and hospitalization, was increased when BEC was above 600 eosinophils/µL. The association between asthma, CRSwNP, and BEC was corroborated by multiple logistic regressions models. (4) Conclusions: We demonstrated the association of having over 600 blood eosinophils/µL with a higher number of hospitalizations and comorbidities (CRSwNP and COPD), which proves that BEC is a highly useful parameter to consider in subjects who present blood eosinophilia.

Shared Decision-Making in Allergen Immunotherapy (AIT) Options Using a Questionnaire for Respiratory Allergic Patients: A Delphi Consensus Study.

Purpose: The objective of this study was to develop and validate a questionnaire, through a Delphi consensus, to be used by allergists in their routine clinical practice to assess the preferences of patients starting allergen immunotherapy (AIT) treatment using an objective approach. Patients and Methods: A Delphi consensus-driven process was used. The scientific committee, composed of 15 allergists, led the study and participated in the preparation of the questionnaire. Two-hundred panelists from different Spanish regions were invited to complete a 16-item questionnaire on a nine-point Likert scale covering six topic blocks. Consensus was achieved if ≥66.6% of panelists reached agreement or disagreement. Results: Of the 200 experts invited to participate in the Delphi process, a total of 195 (97.5%) answered the questionnaire. The panel experts reached a consensus on "agreement" on a total of 12 of the 16 (75.0%) items, covering a total of six categories: (a) patient knowledge (2 questions), (b) barriers to patient adherence (3 questions), (c) patient behavior (4 questions), (d) future actions (3 questions), (e) treatment costs (2 questions), and (f) final patient preferences (2 questions). Conclusion: This Delphi consensus study validated a set of twelve recommended questions for patients objectively assessing their preferences and suitability for the most common AIT options available. The questionnaire intends to assist allergists in making an objective, unconditioned decision regarding the best AIT option for each patient, after informing them about the different routes.

Understanding the Cellular Sources of the Fractional Exhaled Nitric Oxide (FeNO) and Its Role as a Biomarker of Type 2 Inflammation in Asthma.

Fractional exhaled nitric oxide (FeNO) has gained great clinical importance as a biomarker of type 2 inflammation in chronic airway diseases such as asthma. FeNO originates primarily in the bronchial epithelium and is produced in large quantities by the enzyme inducible nitric oxide synthase (iNOS). It should be noted that nitric oxide (NO) produced at femtomolar to picomolar levels is fundamental for respiratory physiology. This basal production is induced in the bronchial epithelium by interferon gamma (IFNγ) via Janus kinases (JAK)/STAT-1 signaling. However, when there is an increase in the expression of type 2 inflammatory cytokines such as IL-4 and IL-13, the STAT-6 pathway is activated, leading to overexpression of iNOS and consequently to an overproduction of airway NO. Increased NO levels contributes to bronchial hyperreactivity and mucus hypersecretion, increases vascular permeability, reduces ciliary heartbeat, and promotes free radical production, airway inflammation, and tissue damage. In asthmatic patients, FeNO levels usually rise above 25 parts per billion (ppb) and its follow-up helps to define asthma phenotype and to monitor the effectiveness of corticosteroid treatment and adherence to treatment. FeNO is also very useful to identify those severe asthma patients that might benefit of personalized therapies with monoclonal antibodies. In this review, we revised the cellular and molecular mechanisms of NO production in the airway and its relevance as a biomarker of type 2 inflammation in asthma.

Serum microRNAs as Tool to Predict Early Response to Benralizumab in Severe Eosinophilic Asthma.

Severe eosinophilic asthma poses a serious health and economic problem, so new therapy approaches have been developed to control it, including biological drugs such as benralizumab, which is a monoclonal antibody that binds to IL-5 receptor alpha subunit and depletes peripheral blood eosinophils rapidly. Biomarkers that predict the response to this drug are needed so that microRNAs (miRNAs) can be useful tools. This study was performed with fifteen severe eosinophilic asthmatic patients treated with benralizumab, and serum miRNAs were evaluated before and after treatment by semi-quantitative PCR (qPCR). Patients showed a clinical improvement after benralizumab administration. Additionally, deregulation of miR-1246, miR-5100 and miR-338-3p was observed in severe asthmatic patients after eight weeks of therapy, and a correlation was found between miR-1246 and eosinophil counts, including a number of exacerbations per year in these severe asthmatics. In silico pathway analysis revealed that these three miRNAs are regulators of the MAPK signaling pathway, regulating target genes implicated in asthma such as NFKB2, NFATC3, DUSP1, DUSP2, DUSP5 and DUSP16. In this study, we observed an altered expression of miR-1246, miR-5100 and miR-338-3p after eight weeks of benralizumab administration, which could be used as early response markers.

Comparison of impulse oscillometry and spirometry for detection of airway hyperresponsiveness to methacholine, mannitol, and eucapnic voluntary hyperventilation in children.

BACKGROUND: Forced expiratory maneuvers are usually difficult in young children. Impulse oscillometry (IOS) requires no active cooperation, is noninvasive, rapid, and easy to perform. This study aimed to compare IOS indexes and forced expiratory volume in 1 second (FEV1) in children for the assessment of bronchial hyperreactivity to methacholine, mannitol, and eucapnic voluntary hyperventilation (EVH). MATERIALS: Children aged 3-14 years (mean 10.0 ± 3.1) with symptoms suggestive of asthma were recruited. IOS measurements were taken before spirometry. Methacholine, mannitol, and EVH tests were performed without a specific order. RESULTS: We included 190 children, whose mean age was 10.0 ± 3.1 years. Changes in FEV1 correlated significantly with variation in IOS indexes (P < .05). The indexes with the greatest discriminative capacity were Z5, R5, and X5. Optimal cut-offs were: for methacholine tests, ≧22% in R5, ≧82% for reactance area (AX), and ≦41% for X5; for the mannitol test, ≧18% in R5, ≧40% in AX, and ≦21% for X5. In the EVH test, ≧23% for R5, ≧40% for AX, and a fall of 29% for X5. When using the optimal cut-off points obtained from IOS, the mean number of steps and doses required for methacholine and mannitol tests to induce significant bronchoconstriction were significantly lower compared with spirometry ( P < .05). CONCLUSIONS: The effectiveness of R5, X5, and AX indexes were comparable to FEV1 in assessing bronchial obstruction during bronchial challenge testing. Therefore, IOS may be useful in assessing bronchial obstruction in children who cannot reliably perform spirometric maneuvers during bronchial challenge testing.

Asthma diagnosis using integrated analysis of eosinophil microRNAs.

BACKGROUND: Asthma is a syndrome characterized by airway inflammation and obstruction. Due to its heterogeneity, the difficulties in asthma diagnosis and treatment make the discovery of new biomarkers a focus of research. So, we determined the differential miRNA expression of eosinophils between healthy and asthmatic patients and to establish a differentially expressed miRNA profile detectable in sera for use as biomarker. METHODS: MicroRNAs from peripheral eosinophils from healthy and asthmatic subjects were isolated and analyzed by next-generation sequencing and confirmed by quantitative PCR in 29 asthmatics and 10 healthy individuals. The levels of serum miRNAs were performed by quantitative PCR in 138 asthmatics and 39 healthy subjects. Regression analysis and Random Forest models were performed. RESULTS: We found a set of miRNAs whose expression differs between eosinophils from asthmatics and healthy subjects. These miRNAs can classify asthmatics into two clusters that differed in the number of eosinophils and periostin concentration in serum. Some of these miRNAs were also confirmed in sera, as miR-185-5p which discriminates asthmatics from healthy subjects. Together with other two miRNAs, miR-185-5p allowed us to create a logistic regression model to discriminate better both conditions and a Random Forest model that can even sort the asthmatics into intermittent, mild persistent, moderate persistent, and severe persistent asthma. CONCLUSION: Our data show that miRNAs profile in eosinophils can be used as asthma diagnosis biomarker in serum and that this profile is able to rank asthma severity.

Exosomes from eosinophils autoregulate and promote eosinophil functions.

Eosinophils are able to secrete exosomes that have an undefined role in asthma pathogenesis. We hypothesized that exosomes released by eosinophils autoregulate and promote eosinophil function. Eosinophils of patients with asthma (n = 58) and healthy volunteers (n = 16) were purified from peripheral blood, and exosomes were isolated and quantified from eosinophils of the asthmatic and healthy populations. Apoptosis, adhesion, adhesion molecules expression, and migration assays were performed with eosinophils in the presence or absence of exosomes from healthy and asthmatic individuals. Reactive oxygen species (ROS) were evaluated by flow cytometry with an intracellular fluorescent probe and nitric oxide (NO) and a colorimetric kit. In addition, exosomal proteins were analyzed by mass spectrometry. Eosinophil-derived exosomes induced an increase in NO and ROS production on eosinophils. Moreover, exosomes could act as a chemotactic factor on eosinophils, and they produced an increase in cell adhesion, giving rise to a specific augmentation of adhesion molecules, such as ICAM-1 and integrin α2. Protein content between exosomes from healthy and asthmatic individuals seems to be similar in both groups. In conclusion, we found that exosomes from the eosinophils of patients with asthma could modify several specific eosinophil functions related to asthma pathogenesis and that they could contribute fundamentally to the development and maintenance of asthma.

New developments in work-related asthma.

INTRODUCTION: Work-related asthma includes two subtypes: occupational asthma or asthma caused by specific agents (sensitizers or irritants) in the workplace, and work-exacerbated asthma or pre-existing asthma worsened by workplace exposures. Areas covered: This review provides an update on the definitions and the clinical features of the different work-related asthma subtypes as well as new insights into their etiology and the pathophysiological mechanisms involved. The diagnosis of work-related asthma should be made on objective basis using a constellation of clinical, physiologic and allergologic tests. Specific inhalation challenge with the suspected occupational agent(s) remains as the reference standard for diagnosis. A literature search was performed using the following terms: work-related asthma, occupational asthma, work-exacerbated asthma, irritant-induced asthma and etiological agents. Expert commentary: Studies focusing on the biological effects and mechanisms of environmental exposures in the development of sensitizer-induced or irritant-induced asthma in various workplace settings are of greatest interest. An integrative approach that combines clinical parameters with component-resolved diagnosis as well as inflammatory biomarkers appears to be very promising. Occupational allergy provides a good opportunity to understand the complex relationships between exposure to allergens in the workplace, interaction with genes and the co-exposures to other factors in the working environment.

SOCS3 silencing attenuates eosinophil functions in asthma patients.

Eosinophils are one of the key inflammatory cells in asthma. Eosinophils can exert a wide variety of actions through expression and secretion of multiple molecules. Previously, we have demonstrated that eosinophils purified from peripheral blood from asthma patients express high levels of suppressor of cytokine signaling 3 (SOCS3). In this article, SOCS3 gene silencing in eosinophils from asthmatics has been carried out to achieve a better understanding of the suppressor function in eosinophils. SOCS3 siRNA treatment drastically reduced SOCS3 expression in eosinophils, leading to an inhibition of the regulatory transcription factors GATA-3 and FoxP3, also interleukin (IL)-10; in turn, an increased STAT3 phosphorilation was observed. Moreover, SOCS3 abrogation in eosinophils produced impaired migration, adhesion and degranulation. Therefore, SOCS3 might be regarded as an important regulator implicated in eosinophil mobilization from the bone marrow to the lungs during the asthmatic process.

Exosome secretion by eosinophils: A possible role in asthma pathogenesis.

BACKGROUND: Eosinophils secrete several granules that are involved in the propagation of inflammatory responses in patients with pathologies such as asthma. OBJECTIVE: We hypothesized that some of these granules are exosomes, which, when transferred to the recipient cells, could modulate asthma progression. METHODS: Eosinophils were purified from peripheral blood and cultured with or without IFN-γ or eotaxin. Multivesicular bodies (MVBs) in eosinophils were studied by using fluorescence microscopy, transmission electron microscopy (TEM), and flow cytometry. Exosome secretion was measured and exosome characterization was performed with TEM, Western blotting, and NanoSight analysis. RESULTS: Generation of MVBs in eosinophils was confirmed by using fluorescence microscopy and flow cytometry and corroborated by means of TEM. Having established that eosinophils contain MVBs, our aim was to demonstrate that eosinophils secrete exosomes. To do this, we purified exosomes from culture medium of eosinophils and characterized them. Using Western blot analysis, we demonstrated that eosinophils secreted exosomes and that the discharge of exosomes to extracellular media increases after IFN-γ stimulation. We measured exosome size and quantified exosome production from healthy and asthmatic subjects using nanotracking analysis. We found that exosome production was augmented in asthmatic patients. CONCLUSION: Our findings are the first to demonstrate that eosinophils contain functional MVBs and secrete exosomes and that their secretion is increased in asthmatic patients. Thus exosomes might play an important role in the progression of asthma and eventually be considered a biomarker.

Nasal response in patients with diisocyanate asthma.

BACKGROUND: To date, no studies have assessed nasal and bronchial response to diisocyanates during specific inhalation challenges (SIC). OBJECTIVES: This study was performed to assess nasal response during SIC with diisocyanates (nasal and oral breathing) in patients with suspected occupational asthma due to these agents. METHODS: Fourteen patients with suspected clinical history of diisocyanate-induced asthma were challenged with diisocynates in a 7m3 chamber. Nasal response testing during challenges was assessed by acoustic rhinometry, peak nasal inspiratory flow (PNIF), and visual analog scale (VAS), alongside bronchial responses. RESULTS: Eleven patients had a significant asthmatic response to diisocyanates. None reported clear work-related nasal symptoms. In patients with positive bronchial response to diisocyanates, nasal mean minimal cross-sectional area (MCA) decreased by 26.9%, nasal volume at 5 cm decreased by 33.5%, and PNIF decreased by 28.3%, all from baseline. A positive nasal response was elicited in 45%, 54%, and 45% of patients, respectively. A significant increase in VAS was observed in 4 patients. Three patients with negative bronchial response had a negative nasal response. CONCLUSION: SIC revealed an objective nasal response in around 50% of patients with occupational asthma due to diisocyanates, in spite of the fact that none of them reported work-related nasal symptoms. The clinical significance of this finding is a poor association between nasal symptoms at work and an objective nasal response during positive SIC with diisocyanates.

Distinctive bronchial inflammation status in athletes: basophils, a new player.

The aim of the study was to establish bronchial inflammation status and to measure eicosanoids in sputum obtained from active elite athletes. A total of 68 subjects were enrolled. Twelve were non-athletes and non-asthmatic (NAtNAs), 21 non-athlete asthmatics (NAtAs), 11 athlete non-asthmatics (AtNAs), and 24 athletes with asthma (AtAs) with positive indirect or direct bronchial challenges. Induced sputum was used to measure cells and eicosanoids. Sputum differential cell counts in all the subject groups revealed eosinophilia with the exception of NAtNAs control subjects. Athletes with and without diagnosed asthma showed a significant increase in bronchial epithelial cells and lymphocytes present in their sputum. Also, flow cytometry revealed that a significantly higher number of basophils were present in sputum from athletes (without and with asthma) when compared with non-athletes (without and with asthma). Asthmatic athletes and non-athletes showed a higher increase in LTC(4) levels and PGE(2) metabolites in sputum when compared with healthy controls. The present study identifies basophils as a new player present in athletes bronchial inflammation defining athlete status and not necessarily associated with exercise-induced bronchoconstriction.

Suppressors of cytokine signaling 3 expression in eosinophils: regulation by PGE2 and Th2 cytokines.

Asthma and nonasthmatic eosinophilic bronchitis (NAEB) are respiratory disorders characterized by a predominance of Th2 cells and eosinophilic inflammation. Suppressors of cytokine signaling (SOCS) proteins play an important role in Th2-mediated allergic responses through control of the balance between Th1 and Th2 cells, particularly, SOCS3 and SOCS5. The aim of this study was to analyze SOCS expression in human peripheral blood eosinophils from patients with asthma, NAEB and healthy controls. SOCS expression in eosinophils from subjects was demonstrated by different techniques. Results showed that expression of SOCS3 in eosinophils and CD4 T cells from patients was higher than in healthy subjects. In addition, we demonstrated that prostaglandin E2 (PGE2) and Th2 cytokines are able to upregulate SOCS3 production in eosinophils and attenuate its degranulation. In conclusion, eosinophils are able to transcribe and translate SOCS3 protein and can contribute to the regulation of the Th1/Th2 balance through SOCS3 production.