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Allergic asthma; Eosinophil subsets; Induced sputum Asma alérgica; Subconjuntos de eosinófilos; Esputo inducido Asma al·lèrgica; Subconjunts d'eosinòfils; Esput induït Two subsets of eosinophils have been described: resident eosinophils with homeostatic functions (rEOS) in healthy subjects and in patients with nonallergic eosinophilic asthma, and inflammatory eosinophils (iEOS) in blood and lung samples from patients with allergic asthma. We explored if it would be possible to identify different subsets of eosinophils using flow cytometry and the gating strategy applied to induced sputum. We conducted an observational cross-sectional single-center study of 62 patients with persistent allergic asthma. Inflammatory cells from induced sputum samples were counted by light microscopy and flow cytometry, and cytokine levels in the supernatant were determined. Two subsets of eosinophils were defined that we call E1 (CD66b-high and CD15-high) and E2 (CD66b-low and CD15-low). Of the 62 patients, 24 were eosinophilic, 18 mixed, 10 paucigranulocytic, and 10 neutrophilic. E1 predominated over E2 in the eosinophilic and mixed patients (20.86% vs. 6.27% and 14.42% vs. 4.31%, respectively), while E1 and E2 were similar for neutrophilic and paucigranulocytic patients. E1 correlated with IL-5, fractional exhaled nitric oxide, and blood eosinophils. While eosinophil subsets have been identified for asthma in blood, we have shown that they can also be identified in induced sputum. This research was supported by the Spanish Allergy and Clinical Immunology Society (SEAIC) by means of a grant awarded in the call of 2017 (reference 17/06) and a BRN—Fundació Pla i Armengol grant in the call of 2018. The funds provided contributed to the acquisition of the material necessary to carry out the study, but the collaborating entities had no role in the analysis or interpretation of the results.

Resumen Introduccion Aunque las guias de practica clinica recomiendan un ajuste continuado del tratamiento del asma, reduciendo la medicacion de mantenimiento cuando se alcanza el control (step-down), existen pocos estudios de practica clinica habitual orientados a recabar informacion de los factores que condicionan su fracaso. Objetivo Determinar los factores que condicionan en la practica clinica habitual el fracaso del step-down en los pacientes asmaticos moderados-graves controlados con una combinacion de glucocorticoides inhalados/agonistas beta-2 adrenergicos de accion larga. Metodo Estudio multicentrico retrospectivo sobre 374 pacientes con asma moderada-grave controlada con glucocorticoides inhalados/agonistas beta-2 adrenergicos de accion larga en quienes el facultativo indico en 2016 un step-down. Resultados El step-down fracaso en el 41,7%. Los factores relacionados con el fracaso fueron: la mayor edad del paciente (p = 0,006), la presencia de 2 o mas comorbilidades (p = 0,016), el mayor nivel de gravedad (persistente grave vs. persistente moderada) (p 40 anos) (p = 0,045), cuanto mas alto es el escalon terapeutico previo (p = 0,003) y posterior al cambio (p Conclusion En condiciones de practica clinica habitual el step-down fracasa en un porcentaje alto de pacientes y se recomienda indicarlo cuando el paciente lleve mas de 6 meses controlado.

Although the clinical practice guidelines recommend continuous adjustment of asthma treatment and reducing the maintenance drugs when achieving control (step-down), there are few studies of standard clinical practice aimed at collecting information on the factors that determine step-down failure.To determine the factors that determine step-down failure in standard clinical practice of patients with moderate-severe asthma controlled by a combination of inhaled glucocorticoids and long-acting beta agonists.A multicentre retrospective study included 374 patients with moderate-severe asthma controlled with inhaled glucocorticoids and long-acting beta agonists for whom the physician indicated a step-down in 2016.The step-down failed in 41.7% of the patients. The following factors were related to failure: greater patient age (P=.006), presence of at least 2 comorbidities (P=.016), greater severity level (severe persistent vs. moderate persistent) (P.001), greater age at diagnosis (40 years) (P=.045), the higher the therapeutic step before (P=.003) and after the change (P.001), the shorter the time of improvement/control prior to the change (P=.019), lower FEVIn standard clinical practice conditions, step-down fails in a high percentage of patients, and the suggestion is to indicate step-down when the patient has had more than 6 months of disease control.

Introduction Asthma with airway mucus hypersecretion is an inadequately characterized variant of asthma. While several studies have reported that hypersecreting patients may carry genetic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, many of those studies have been questioned for their numerous limitations and contradictory results. Objectives (1) To determine the presence of genetic variants of the CFTR gene in patients with asthma with and without airway mucus hypersecretion. (2) To identify the clinical, inflammatory and functional characteristics of the asthma phenotype with airway mucus hypersecretion. Method Comparative multicentre cross-sectional descriptive study that included 100 patients with asthma (39 hypersecretors and 61 non-hypersecretors). Asthmatic hypersecretion was defined as the presence of cough productive of sputum on most days for at least 3 months in 2 successive years. The patients were tested for fractional exhaled nitric oxide, spirometry, induced sputum cell count, total immunoglobulin E (IgE), peripheral blood eosinophil count, C-reactive protein, blood fibrinogen and blood albumin and underwent a skin prick test. Asthma control and quality of life were assessed by the Asthma Control Test and Mini Asthma Quality of Life questionnaires, respectively. Blood DNA samples were collected from the patients and next-generation sequencing using a MiSeq sequencer and the Illumina platform was used for the CFTR gene analysis. Results Genetic differences were observed in the c.1680-870T>A polymorphism of the CFTR gene, significantly more evident in hypersecretors than in non-hypersecretors: 78.94% vs. 59.32% in the majority allele and 21.05% vs. 40.67% in the minority allele (p = 0.036). Clinically, asthma hypersecretors compared to non-hypersecretors were older (57.4 years vs. 49.4 years; p = 0.004); had greater asthma severity (58.9% vs. 23.7%; p = 0.005); experienced greater airway obstruction (FEV1/FVC% 64.3 vs. 69.5; p = 0.041); had poorer asthma control (60% vs. 29%; p = 0.021); had lower IgE levels (126.4 IU/mL vs. 407.6 IU/mL; p = 0.003); and were less likely to have a positive prick test (37.5% vs. 68.85%; p = 0.011). Conclusion The results suggest that patients with asthma and with mucus hypersecretion (1) may have a different phenotype and disease mechanism produced by an intronic polymorphism in the CFTR gene (NM_000492.3:c.1680-870T>A), and (2) may have a poorer clinical outcome characterized by severe disease and poorer asthma control with a non-allergic inflammatory phenotype.

Sputum induction (SI) is the gold standard approach to the non-invasive study of airway inflammation. The differential count of inflammatory cells for SI allows patients with asthma to be classified according to inflammatory phenotypes and predicted therapeutic responses. Since SI involves the generation of aerosols, there is a need to establish a protocol to ensure biosafety in clinical practice during the current COVID-19 pandemic. The multidisciplinary consensus on SI described in this article was developed by 22 experts in SI from different Spanish hospitals who drew on available scientific evidence in achieving consensuated opinions, compiled by means of an electronic survey. We hope that these unified criteria and recommendations will guide health professionals in implementing SI sampling and processing procedures as safely as possible during the COVID-19 pandemic.

Asthma with bronchial hypersecretion is a type of asthma that is poorly studied. Its pathogenesis is not well understood, but is probably related to innate impaired immunity, particularly with toll-like receptors (TLRs) and secretory mucins (MUC). Objectives 1) Define the clinical and inflammatory phenotype of asthma with bronchial hypersecretion of mucus. 2) Compare the type of mucin present in induced sputum (IS) of patients with and without bronchial hypersecretion. 3) Determine the expression of TLRs in IS and blood of asthmatics with and without bronchial hypersecretion. Materials and methods Cross-sectional study which included 43 non-smoking asthmatic patients without bronchiectasis, 19 with bronchiectasis, and 24 without bronchial hypersecretion. All patients underwent the following: IS, spirometry, fractional exhaled nitric oxide, prick test, total immunoglobulin E (IgE), and blood albumin. Analysis of mucins was determined by ELISA and expression of TLR2 and TLR4 by flow cytometry. The level of asthma control was determined by the Asthma Control Test (ACT) questionnaire and quality of life was assessed by the reduced version of the Asthma Quality of Life Questionnaire (mini-AQLQ). Results Asthmatics with bronchial hypersecretion were significantly older (62.6 years vs 48.5 years; p=0.02); had greater severity (persistent severe asthma 94.7% vs 29.2%; p=0.000); a higher proportion of nasal polyposis (36.8% vs 8.3%; p=0.022); less control of asthma (73.7% vs 8.3%; p=0,000); a higher proportion of asthma with negative prick test (68.4% vs 16.6%; p=0.001), and lower levels of IgE (113.4 IU/mL vs 448 IU/mL; p=0.007), compared with asthmatics without bronchial hypersecretion. Significant differences were observed neither in the expression of TLRs 2 and 4 in inflammatory cells of IS or peripheral blood, nor in the expression of mucins between both groups. Conclusion Asthma patients with bronchial hypersecretion have more severe and uncontrolled disease, with poor quality of life as well as a non-allergic inflammatory phenotype. Within the mechanisms involving these differences, it does not appear that mucins and TLRs play an important role., Video abstract

Background Most patients with nonallergic asthma have normal serum immunoglobulin E (IgE) levels. Recent reports suggest that total and aeroallergen-specific IgE levels in induced sputum may be higher in nonallergic asthmatics than in healthy controls. Our objective is to compare total and dust-mite specific (Der p 1) IgE levels in induced sputum in allergic and nonallergic asthmatics and healthy controls. Methods Total and Der p 1-specific IgE were measured in induced sputum (ImmunoCAP immunoassay) from 56 age- and sex-matched asthmatics (21 allergic, 35 nonallergic) and 9 healthy controls. Allergic asthma was defined as asthma with a positive prick test and/or clinically-significant Der p 1-specific serum IgE levels. Results Patients with allergic asthma presented significantly higher total and Der p 1-specific serum IgE levels. There were no significant between-group differences in total sputum IgE. However, Der p 1-specific sputum IgE levels were significantly higher (p = 0.000) in the allergic asthmatics, but without differences between the controls and nonallergic asthmatics. Serum and sputum IgE levels were significantly correlated, both for total IgE (rho = 0.498; p = 0.000) and Der p 1-specific IgE (rho, 0.621; p = 0001). Conclusions Total IgE levels measured in serum and induced sputum are significantly correlated. No significant differences were found between the differents groups in total sputum IgE. Nevertheless, the levels of Der p 1-specific sputum IgE levels were significantly higher in the allergic asthmatics, but without differences between the controls and nonallergic asthmatics. Probably due to the lack of sensitivity of the test used, but with the growing evidence for local allergic reactions better methods are need to explore its presence. The Clinical Trials Identifier for this project is NCT03640936.

BackgroundContinuous positive airway pressure (CPAP) in asthma patients with concomitant obstructive sleep apnea syndrome (OSAS) seems to have a favorable impact on asthma, but data are inconsistent due to methodological limitations of previous studies. MethodsProspective, multicenter study. We examined asthma outcomes after 6 months of CPAP in 99 adult asthma patients (mean age 57 years) with OSAS (respiratory disturbance index 20). Asthma control and quality of life were assessed with the Asthma Control Questionnaire (ACQ) and the Mini Asthma Quality of Life Questionnaire (MiniAQLQ), respectively. Data were analyzed by intention-to-treat basis. ResultsThe mean SD score of the ACQ decreased from 1.39 +/- 0.91 at baseline to 1.0 +/- 0.78 at 6 months (P = 0.003), the percentage of patients with uncontrolled asthma from 41.4% to 17.2% (P = 0.006), and the percentage of patients with asthma attacks in the 6 months before and after treatment from 35.4% to 17.2% (P = 0.015). The score of the mAQLQ increased from 5.12 +/- 1.38 to 5.63 +/- 1.17 (P = 0.009). There were also significant improvements in symptoms of gastroesophageal reflux and rhinitis, bronchial reversibility, and exhaled nitric oxide values (all P < 0.05). No significant changes were observed in drug therapy for asthma or their comorbidities nor in the patients' weight. ConclusionsAsthma control (both actual and future risk), quality of life, and lung function improved after starting continuous positive airway pressure in asthmatics with moderate to severe obstructive sleep apnea syndrome., This work was supported by a grant from the Spanish Society of Pneumology and Thoracic Surgery (SEPAR): 'Convocatoria Extraordinaria PII 2010-PII de Asma' (Barcelona, Spain, 2011).

Background: Emerging data suggest that innate immunity may play a role in asthma, particularly the toll-like receptors (TLRs). Some studies pointed to an involvement of TLRs 2 and 4 in the pathogenesis of allergic asthma, and other studies related TLRs to IgE. However, there are not any studies that have comprehensively evaluated the expression of TLRs 2 and 4 in inflammatory cells, in peripheral blood and induced sputum specimens from asthmatic patients, according to their total serum IgE. Methods: We studied 44 asthmatic patients (15 with high total serum IgE and 29 with normal total serum IgE). On a single visit, all patients underwent: induced sputum, pulmonary function tests, determination of exhaled nitric oxide fraction, venipuncture for blood analysis and skin prick allergy tests. The induced sputum cellularity was analyzed by flow cytometry, where expression of TLRs 2 and 4 was studied using fluorochrome-conjugated monoclonal antibodies. Results: Asthmatic patients with high total serum IgE showed, a higher percentage of macrophages expressing TLR4 (42.99 % +/- 22.49) versus asthmatic patients with normal total serum IgE (28.84 % +/- 15.16) (P = 0.048). Furthermore, we observed a correlation (but weak) between the percentage of macrophages expressing TLR4 in induced sputum and the total serum IgE level (R = 0.314; P = 0.040). Conclusion: Asthmatic subjects with high total serum IgE show increased macrophage expression of TLR4 in induced sputum. This outcome may result from a link between innate immunity and IgE-mediated, adaptive immune responses in asthma, and point to TLR4 as a potential therapeutic target.

La tesis doctoral que presento es el resultado del trabajo realizado en el Servicio de Neumología del Hospital de la Santa Creu i Sant Pau de Barcelona y del Institut de Recerca Biomèdica de Sant Pau (IIB Sant Pau), siguiendo las normativas de la Universitat Autònoma de Barcelona para su presentación como compendio de publicaciones. El estudio de la inflamación bronquial en el asma, se ha convertido en una herramienta valiosa para el diagnóstico, control y predicción de respuestas terapéuticas. En los últimos años ha aumentado el interés en los métodos que permitan evaluar de una forma no invasiva la inflamación de la vía aérea. Entre los métodos no invasivos descritos usados en la práctica clínica para evaluar la inflamación de la vía aérea figuran: el recuento de células inflamatorias en el esputo inducido y la fracción exhalada de óxido nítrico (FeNO). La temperatura del aire exhalado y la nariz electrónica se plantean como nuevas herramientas de medición de la inflamación bronquial y del control del asma. La línea de investigación desarrollada tuvo como objetivo fundamental mejorar los conocimientos sobre los fenotipos inflamatorios del asma a través de métodos no invasivos. Está basada en tres proyectos. El primero, es un proyecto clínico cuyos resultados muestran que existen un alto porcentaje de pacientes con disociación entre los resultados de la FeNO y de los eosinófilos en el esputo inducido y que cursan con características clínicas e inflamatorias diferenciales. Los otros dos proyectos permiten conocer la utilidad en el asma de dos nuevos métodos no invasivos como lo son, la temperatura del aire exhalado (TAE) y el reconocimiento de patrones de compuestos orgánicos volátiles mediante la “nariz electrónica”. Estos trabajos aportaron como resultados principales que en el caso de la TAE, no parece que éste sea un método que proporcione una información clínica útil puesto que no se encontró ninguna correlación entre este método y el grado de control del asma, la gravedad de la enfermedad, la obstrucción bronquial, o la inflamación bronquial. Por otro lado, los resultados del tercer estudio sí que fueron alentadores, puesto que de manera significativa, el uso de la nariz electrónica en un entorno clínico habitual permitió discriminar con fiabilidad los diferentes fenotipos inflamatorios bronquiales en pacientes con asma. Así pues, los resultados de esta tesis sirven como fundamento del estudio y aplicabilidad de diversos métodos no invasivos en el asma., This doctoral thesis is the result of the work done in the Service of Respiratory Medicine of the Hospital de la Santa Creu i Sant Pau in Barcelona and the Institute of Biomedical Research of Sant Pau (IIB Sant Pau), following the regulations of the Universitat Autònoma de Barcelona. The study of bronchial inflammation in asthma has become a valuable tool for its diagnosis, monitoring and prediction of therapeutic responses. In recent years, there has been an increased interest in methods of noninvasive evaluation of the airway inflammation. The described non-invasive methods used in the clinical practice to assess airway inflammation include the inflammatory cell counts in induced sputum and the fractional exhaled nitric oxide (FeNO). The exhaled breath temperature and the electronic nose device are considered to be new tools for measuring airway inflammation and control of asthma. The line of the developed research had as a main goal to improve the knowledge of inflammatory phenotypes of asthma through non-invasive methods. It is based on three projects. The first (1) is a clinical project that shows a high percentage of patients with dissociation between the results of the FeNO and eosinophils in induced sputum, presenting clinical and inflammatory differential characteristics. The other two projects provide an insight into the utility of two non-invasive diagnostic methods: 2) the exhaled breath temperature (EBT) and 3) the recognition of the patterns formed by organic volatile compounds using the electronic nose device. The second study does not support the usefulness of the EBT plateau, because no correlation was found between EBT and control of asthma, severity of disease, bronchial obstruction or bronchial inflammation. Furthermore, the results of the third study were encouraging since the using of an e-nose device in a regular clinical setting can reliably discriminate different inflammatory asthma phenotypes among patients with persistent asthma. Thus, the results of this thesis disclosed the applicability of various non-invasive methods performed in routine clinical practice.

Background: The measurement of fractional nitric oxide concentration in exhaled breath (FeNO), a noninvasive indicator of airway inflammation, remains controversial as a tool to assess asthma control. Guidelines currently limit asthma control assessment to symptom and spirometry based appraisals such as the Asthma Control Questionnaire-7 (ACQ-7). We aimed at determining whether adding FeNO to ACQ-7 improves current asthma clinical control assessment, through enhanced detection of not well controlled asthma. Methods: Asthmatic subjects, classified as not well controlled as per ACQ-7 on regular clinical practice, were included in a prospective, multicenter fashion, and had their maintenance treatment adjusted on visit 1. On follow-up (visit 2) four weeks later, the subjects were reevaluated as controlled or not well controlled using ACQ-7 versus a combination of FeNO and ACQ-7. Results: Out of 381 subjects enrolled, 225 (59.1%) had not well controlled asthma on visit 2 as determined by ACQ-7, and 264 (69.3%) as per combined FeNO and ACQ-7. The combination of FeNO to ACQ-7 increased by 14.8% the detection of not well controlled asthma following maintenance therapy adjustment. Conclusions: The addition of FeNO to ACQ-7 increased the detectability of not well controlled asthma upon adjustment of maintenance therapy. Adding a measure of airway inflammation to usual symptom and spirometry based scores increases the efficacy of current asthma clinical control assessment.