An integrated metabo-lipidomics profile of induced sputum for the identification of novel biomarkers in the differential diagnosis of asthma and COPD.

BACKGROUND: Due to their complexity and to the presence of common clinical features, differentiation between asthma and chronic obstructive pulmonary disease (COPD) can be a challenging task, complicated in such cases also by asthma-COPD overlap syndrome. The distinct immune/inflammatory and structural substrates of COPD and asthma are responsible for significant differences in the responses to standard pharmacologic treatments. Therefore, an accurate diagnosis is of central relevance to assure the appropriate therapeutic intervention in order to achieve safe and effective patient care. Induced sputum (IS) accurately mirrors inflammation in the airways, providing a more direct picture of lung cell metabolism in comparison to those specimen that reflect analytes in the systemic circulation. METHODS: An integrated untargeted metabolomics and lipidomics analysis was performed in IS of asthmatic (n = 15) and COPD (n = 22) patients based on Ultra-High-Pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS) and UHPLC-tandem MS (UHPLC-MS/MS). Partial Least Squares-Discriminant Analysis (PLS-DA) was applied to resulting dataset. The analysis of main enriched metabolic pathways and the association of the preliminary metabolites/lipids pattern identified to clinical parameters of asthma/COPD differentiation were explored. Multivariate ROC analysis was performed in order to determine the discriminatory power and the reliability of the putative biomarkers for diagnosis between COPD and asthma. RESULTS: PLS-DA indicated a clear separation between COPD and asthmatic patients. Among the 15 selected candidate biomarkers based on Variable Importance in Projection scores, putrescine showed the highest score. A differential IS bio-signature of 22 metabolites and lipids was found, which showed statistically significant variations between asthma and COPD. Of these 22 compounds, 18 were decreased and 4 increased in COPD compared to asthmatic patients. The IS levels of Phosphatidylethanolamine (PE) (34:1), Phosphatidylglycerol (PG) (18:1;18:2) and spermine were significantly higher in asthmatic subjects compared to COPD. CONCLUSIONS: This is the first pilot study to analyse the IS metabolomics/lipidomics signatures relevant in discriminating asthma vs COPD. The role of polyamines, of 6-Hydroxykynurenic acid and of D-rhamnose as well as of other important players related to the alteration of glycerophospholipid, aminoacid/biotin and energy metabolism provided the construction of a diagnostic model that, if validated on a larger prospective cohort, might be used to rapidly and accurately discriminate asthma from COPD.

Stratification of asthma by lipidomic profiling of induced sputum supernatant.

BACKGROUND: Asthma is a chronic respiratory disease with significant heterogeneity in its clinical presentation and pathobiology. There is need for improved understanding of respiratory lipid metabolism in asthma patients and its relation to observable clinical features. OBJECTIVE: We performed a comprehensive, prospective, cross-sectional analysis of the lipid composition of induced sputum supernatant obtained from asthma patients with a range of disease severities, as well as from healthy controls. METHODS: Induced sputum supernatant was collected from 211 adults with asthma and 41 healthy individuals enrolled onto the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study. Sputum lipidomes were characterized by semiquantitative shotgun mass spectrometry and clustered using topologic data analysis to identify lipid phenotypes. RESULTS: Shotgun lipidomics of induced sputum supernatant revealed a spectrum of 9 molecular phenotypes, highlighting not just significant differences between the sputum lipidomes of asthma patients and healthy controls, but also within the asthma patient population. Matching clinical, pathobiologic, proteomic, and transcriptomic data helped inform the underlying disease processes. Sputum lipid phenotypes with higher levels of nonendogenous, cell-derived lipids were associated with significantly worse asthma severity, worse lung function, and elevated granulocyte counts. CONCLUSION: We propose a novel mechanism of increased lipid loading in the epithelial lining fluid of asthma patients resulting from the secretion of extracellular vesicles by granulocytic inflammatory cells, which could reduce the ability of pulmonary surfactant to lower surface tension in asthmatic small airways, as well as compromise its role as an immune regulator.

Differential Circular RNA Expression Profiles in Induced Sputum of Patients with Asthma.

INTRODUCTION: circRNA played a role in a variety of diseases. This paper aimed to explore the differentially expressed circRNA in induced sputum cells of asthma patients, so as to provide new potential biomarkers and new ideas for the study of asthma. METHODS: All subjects were from the First Affiliated Hospital of Sun Yat-sen University. Differentially expressed circRNAs of asthma patients were screened by high-throughput sequencing. qRT-PCR was used to verify the expression of differential circRNAs. The association between circRNA and asthma was explored by analyzing the correlation between circRNA and clinical data and some cytokines of asthma patients. The possible ceRNA network was analyzed and predicted by online software, and the expression of each molecule in the network was preliminary verified by qRT-PCR in induced sputum cells and 16HBE cell. RESULTS: We screened a total of 49 circRNAs differentially expressed in asthma patients (including 12 circRNAs with elevated expression and 37 circRNAs with decreased expression), among which has_circSORT1 and has_circSERPINB1 were significantly elevated. Correlation analysis showed that has_circSORT1 was correlated with FeNO, EOS%, IL-17A, IFN-γ, and PC20, and has_circSERPINB1 was correlated with IL-6, IL-17A, IFN-γ, FEV1%, and FVC%. The possible existence of has_circSORT1/has-miR-185-3p/ZNFX1, a ceRNA regulatory network, in induced sputum cells of asthma patients was hypothesized by online software prediction and qRT-PCR in sputum cells and 16HBE. CONCLUSION: Differentially expressed circRNAs existed in induced sputum cells of asthma patients, among which has_circSORT1 and has_circSERPINB1 were significantly upregulated and may be involved in the process of asthma disease, which could be expected to be a potential biomarker for asthma diagnosis. In addition, a ceRNA regulatory network, has_circSORT1/has-miR-185-3p/ZNFX1, may exist in asthma.

TCN1 Expression Is Increased in Asthma.

INTRODUCTION: Asthma is a chronic disease that affects populations worldwide. The purpose of this study was to investigate the expression of TCN1 in sputum and its correlation with inflammation and lung function in asthma. METHODS: We recruited 141 subjects, detected TCN1 mRNA level by quantitative reverse transcription polymerase chain reaction, detected TCN1 protein expression by Western blot, detected TCN1 protein level by enzyme-linked immunosorbent assay, and analyzed the correlation between TCN1 and fraction of exhaled nitric oxide (FeNO), IgE, EOS%, lung functions, and some Th2 cytokines. The diagnostic value of TCN1 was evaluated by receiver operating characteristics curve. The expression of TCN1 was further confirmed by human bronchial epithelial cell in vitro. RESULTS: Compared with the health group, the expression of TCN1 in induced sputum cells increased in asthma group and was correlated with FeNO, IgE, and EOS%. TCN1 level was also elevated in the induced sputum supernatant of asthma patients. The protein level of TCN1 in induced sputum supernatant was correlated with FeNO, IgE and PC-20, forced expiratory volume in the first second (FEV1)%pred, FEV1/FVC, and some cytokines (IL-4, IL-5, IL-10, IL-13, MUC5AC). TCN1 was also differentially expressed in patients with different severity of asthma. Four weeks after ICS treatment, the expression of TCN1 in induced sputum supernatant increased. In vitro, the protein level of TCN1 in human bronchial epithelial cells' supernatant increased after stimulated with IL-4 and IL-13. CONCLUSION: The expression of TCN1 was increased in asthma patients' sputum, and was positively correlated with some inflammatory markers, negatively correlated with lung function. TCN1 may be used as a potential biomarker for the diagnosis and treatment of asthma.

Increased expression of IL1-RL1 is associated with type 2 and type 1 immune pathways in asthma.

BACKGROUND: Asthma is a common chronic airway disease in the world. The purpose of this study was to explore the expression of IL1-RL1 in sputum and its correlation with Th1 and Th2 cytokines in asthma. METHODS: We recruited 132 subjects, detected IL1-RL1 protein level in sputum supernatant by ELISA, and analyzed the correlation between the expression level of IL1-RL1 and fraction of exhaled nitric oxide (FeNO), IgE, peripheral blood eosinophil count (EOS#), and Th2 cytokines (IL-4, IL-5, IL-10, IL-13, IL-33 and TSLP) and Th1 cytokines (IFN-γ, IL-2, IL-8). The diagnostic value of IL1-RL1 was evaluated by ROC curve. The expression of IL1-RL1 was further confirmed by BEAS-2B cell in vitro. RESULTS: Compared with the healthy control group, the expression of IL1-RL1 in sputum supernatant, sputum cells and serum of patients with asthma increased. The AUC of ROC curve of IL1-RL1 in sputum supernatant and serum were 0.6840 (p = 0.0034), and 0.7009 (p = 0.0233), respectively. IL1-RL1 was positively correlated with FeNO, IgE, EOS#, Th2 cytokines (IL-4, IL-5, IL-10, IL-13, IL-33 and TSLP) and Th1 cytokines (IFN-γ, IL-2, IL-8) in induced sputum supernatant. Four weeks after inhaled glucocorticoids (ICS) treatment, the expression of IL1-RL1 in sputum supernatant and serum was increased. In vitro, the expression of IL1-RL1 in BEAS-2B was increased after stimulated by IL-4 or IL-13 for 24 h. CONCLUSION: The expression of IL1-RL1 in sputum supernatant, sputum cells and serum of patients with asthma was increased, and was positively correlated with some inflammatory markers in patients with asthma. IL1-RL1 may be used as a potential biomarker for the diagnosis and treatment of asthma.

Bronchial inflammation biomarker patterns link humoral immunodeficiency with bronchiectasis-related small airway dysfunction.

BACKGROUND: The progression of chronic destructive lung disease in patients with humoral immunodeficiency (ID) and concomitant development of bronchiectasis is difficult to prevent. Lung function tests in these patients typically show bronchial obstruction of the small airways in combination with increased air trapping in the distal airways, which is consistent with small airway dysfunction. OBJECTIVE: The objective was to assess the grade of chronic lower airway inflammation and small airway dysfunction from induced sputum and the corresponding local pro-inflammatory mediator pattern to discriminate patients affected by bronchiectasis-related Small Airway Dysfunction (SAD). METHODS: In a prospective design, 22 patients with ID (14 CVID, 3 XLA, 3 hyper-IgM syndrome, 1 hyper-IgE syndrome and low IgG levels due to treatment with rituximab and 1 SCID after BMT and persistent humoral defect) and 21 healthy controls were examined. Lung function, Fraction Expiratory Nitric Oxide (FeNO) and pro-inflammatory cytokine levels were compared in subsets of patients with (ID + BE) and without bronchiectasis (ID) pre-stratified using high-resolution computed tomography (HRCT) scans and control subjects. RESULTS: Analysis of induced sputum showed significantly increased total cell counts and severe neutrophilic inflammation in ID. The concomitant SAD revealed higher total cell numbers compared to ID. Bronchial inflammation in ID is clearly mirrored by pro-inflammatory mediators IL-1ß, IL-6 and CXCL-8, whilst TNF-α revealed a correlation with lung function parameters altered in the context of bronchiectasis-related Small Airway Dysfunction. CONCLUSIONS: In spite of immunoglobulin substitution, bronchial inflammation was dominated by neutrophils and was highly increased in patients with ID + BE. Notably, the pro-inflammatory cytokines in patients with ID were significantly increased in induced sputum. The context-dependent cytokine pattern in relation to the presence of concomitant bronchiectasis associated with SAD in ID patients could be helpful in delimiting ID patient subgroups and individualizing therapeutic approaches.

Increased SERPINB10 Expression in Induced Sputum Was Associated with Airway Type 2 Inflammation in Asthma.

BACKGROUND: Asthma is a common chronic airway inflammation in the world. The aim of this study was to investigate the expression of SERPINB10 in induced sputum and its correlation with airway type 2 inflammation in asthma. METHODS: We recruited 90 subjects and detected SERPINB10 levels in induced sputum by ELISA and analyzed the correlation between SERPINB10 expression levels and FeNO, eosinophils in peripheral blood, lung function, and Th2 cytokines (IL-4, IL-5, and IL-13). RESULTS: The levels of SERPINB10 in induced sputum in asthmatic patients were higher than healthy controls. SERPINB10 levels in induced sputum were positively correlated with FeNO (r = 0.4620, p < 0.0001) and eosinophils in peripheral blood (r = 0.2500, p = 0.0218) and negatively correlated with FEV1 (%predicted) (r = -0.4161, p < 0.0001) and FEV1/FVC% (r = -0.4383, p < 0.0001). SERPINB10 levels were correlated with Th2 cytokines IL-4 (r = 0.6274, p < 0.0001), IL-5 (r = 0.5166, p < 0.0001), and IL-13 (r = 0.5212, p = 0.0003) in asthma. CONCLUSIONS: SERPINB10 levels in induced sputum of asthmatic patients were significantly increased and correlated with asthmatic airway type 2 inflammation. Induced sputum SERPINB10 may be a signature protein for type 2 high asthma and may be a potential target for airway eosinophilic inflammation in asthma.

Correlation between Increased Monocyte Chemotactic Protein 1 Level and Lung Function in Patients with Asthma.

BACKGROUND: The altered expression of monocyte chemotactic protein 1 (MCP1) in bronchoalveolar fluid was observed in patients and animal models of allergic asthma. However, the correlation between induced sputum MCP1 level and asthma clinical features remains poorly understood. OBJECTIVE: This study aims to explore the relationship of MCP1 protein expression in induced sputum with Th2 inflammation and asthma clinical features. METHODS: MCP1 protein expression in induced sputum and serum was detected by ELISA in patients with asthma, and its correlation with Th2 inflammation and lung function was analyzed. The effect of inhaled corticosteroids (ICSs) on MCP1 expression was also evaluated. RESULTS: The MCP1 level in induced sputum (p = 0.0006) and serum (p = 0.0035) was markedly increased and negatively correlated with forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC)% (r = -0.3674, p = 0.0001) and PC-20 (r = -0.5746, p < 0.0001) in patients with asthma. The area under the curve (AUC) of MCP1 level receiver operating characteristic curve in induced sputum and serum was 0.7134 (p = 0.0007) and 0.7589 (p = 0.0042), respectively. The patients with asthma were grouped into four according to their induced sputum MCP1 level and Th2 signature categories (Th2Hi MCP1Hi, Th2Hi MCP1Low, Th2Low MCP1Hi, and Th2Low MCP1Low). The Th2Low MCP1Hi subgroup had the lowest FEV1/FVC% and histamine concentration required to cause a 20% decline in FEV1. After 4 weeks of ICS treatment, the MCP1 levels in induced sputum and serum were significantly reduced. CONCLUSIONS: The MCP1 level in induced sputum and serum increased in patients with asthma but decreased after ICS treatment. The Th2Low MCP1Hi subgroup had the worst lung function and highest airway hyperresponsiveness. The combination of MCP1 level in induced sputum and Th2 inflammation defines a new phenotype that can be used to predict lung function and treatment response in patients with asthma.

Elevated CEACAM5 Levels in Patients with Asthma.

INTRODUCTION: Asthma is a common chronic respiratory disease. This study aimed to explore the expression level of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in induced sputum supernatant, induced sputum cells, and serum of asthma patients. METHODS: The protein levels of CEACAM5 in induced sputum supernatant and serum were detected by enzyme-linked immunosorbent assay. The expression of CEACAM5 in induced sputum cells was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). We analyzed the correlations between CEACAM5 expression and the clinical characteristics (FeNO and IgE) of asthma. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of CEACAM5 in asthma. The expression level of CEACAM5 in 16HBE and BEAS-2B cells was detected by qRT-PCR. RESULTS: The expression of CEACAM5 in induced sputum supernatant, induced sputum cells, and serum of asthma patients was significantly upregulated. Asthma patients with high CEACAM5 expression in induced sputum supernatant had higher levels of FeNO, IgE, and IL-13. The expression levels of CEACAM5 in induced sputum supernatant and induced sputum cells were positively correlated with FeNO and IgE. The ROC curve showed that CEACAM5 had a good diagnostic value in asthma. CEACAM5 expression was upregulated in BEAS-2B and 16HBE cells after IL-4 or IL-13 stimulation for 48 h. CONCLUSION: The expression levels of CEACAM5 in induced sputum supernatant, induced sputum cells, and serum of asthma patients were significantly increased. CEACAM5 may be involved in eosinophilic inflammation of asthma and may be used as a diagnostic biomarker and therapeutic target of asthma.

Elevated CXCL14 in Induced Sputum Was Associated with Eosinophilic Inflammation and Airway Obstruction in Patients with Asthma.

INTRODUCTION: CXCL14 involved in inflammatory processes was upregulated in the asthma expression profile datasets in our pilot study. However, the expression of CXCL14 in induced sputum and its potential clinical role in asthma were poorly reported. OBJECTIVE: We sought to detect CXCL14 expression in airway epithelium and induced sputum cells of asthma and explore its potential clinical implications. METHODS: The expression of CXCL14 in asthma was analyzed using R software based on multiple microarray datasets, including GSE43696, GSE63142, GSE67940, and GSE76262. Subsequent verification of the CXCL14 expression pattern in induced sputum and bronchial epithelium cells was performed by qRT-PCR and ELISA. Besides, the correlations between CXCL14 and eosinophilic inflammation indicators (FeNO, EOS#, and IgE), Th2 signature genes (SERPINB2, POSTN, and CLCA1), inflammatory cytokines (IL-4, IL-5, IL-10, IL-13, IL-25, IL-33, TSLP, IL-8, IL-17A, IFN-γ, and IL-2), and airway obstruction indicators (pulmonary function and mucin secretion) were further explored. RESULTS: The expression of CXCL14 in epithelium and sputum cells was upregulated in asthma and positively correlated with clinical eosinophilic indicators. The protein levels of CXCL14 were positively associated with Th2 signature genes (SERPINB2, POSTN, and CLCA1) and Th2 cytokines (IL-4, IL-5, IL-10, IL-13, IL-25, IL-33, and TSLP). Increased expression of CXCL14 was also observed in BEAS-2B cells stimulated by the cytokine IL-4. Furthermore, the expression of CXCL14 was positively correlated with MUC5AC secretion and negatively associated with pulmonary function. CONCLUSIONS: Upregulated CXCL14 in asthma was positively correlated with inflammatory indicators and negatively correlated with pulmonary function, which indicated that upregulated CXCL14 might act as a pathogenic gene through involvement in Th2 inflammation in asthma.

Diagnostic stewardship aiming at expectorated or induced sputum promotes microbial diagnosis in community-acquired pneumonia.

PURPOSE: Studies on aetiology of community-acquired pneumonia (CAP) vary in terms of microbial sampling methods, anatomical locations, and laboratory analyses, since no gold standard exists. In this large, multicentre, retrospective, regional study from Norway, our primary objective was to report the results of a strategic diagnostic stewardship intervention, targeting diagnostic yield from lower respiratory tract sampling. The secondary objective was to report hospitalized CAP aetiology and the diagnostic yield of various anatomical sampling locations. METHODS: Medical records from cases diagnosed with hospitalized CAP were collected retrospectively from March throughout May for three consecutive years at six hospitals. Between year one and two, we launched a diagnostic stewardship intervention at the emergency room level for the university teaching hospital only. The intervention was multifaceted aiming at upscaling specimen collection and enhancing collection techniques. Year one at the interventional hospital and every year at the five other emergency hospitals were used for comparison. RESULTS: Of the 1280 included cases of hospitalized CAP, a microbiological diagnosis was established for 29.1% among 1128 blood cultures and 1444 respiratory tract specimens. Blood cultures were positive for a pathogenic respiratory tract microbe in 4.9% of samples, whereas upper and lower respiratory tract samples overall provided a probable microbiological diagnosis in 21.3% and 47.5%, respectively. Expectorated or induced sputum overall provided aetiology in 51.7% of the samples. At the interventional hospital, the number of expectorated or induced sputum samples were significantly increased, and diagnostic yield from expectorated or induced sputum was significantly enhanced from 41.2 to 62.0% after the intervention (p = 0.049). There was an over-representation of samples from the interventional hospital during the study period. Non-typeable Haemophilus influenza and Streptococcus pneumoniae accounted for 25.3% and 24.7% of microbiologically confirmed cases, respectively. CONCLUSION: Expectorated or induced sputum outperformed other sampling methods in providing a reliable microbiological diagnosis for hospitalized CAP. A diagnostic stewardship intervention significantly improved diagnostic yield of lower respiratory tract sampling.

Automated cell differential count in sputum is feasible and comparable to manual cell count in identifying eosinophilia.

INTRODUCTION: Cell differential count (CDC) of induced sputum is considered the gold standard for inflammatory phenotyping of asthma but is not implemented in routine care due to its heavy time- and staff demands. Digital Cell Morphology is a technique where digital images of cells are captured and presented preclassified as white blood cells (neutrophils, eosinophils, lymphocytes, macrophages, and unidentified) and nonwhite blood cells for review. With this study, we wanted to assess the accuracy of an automated CDC in identifying the key inflammatory cells in induced sputum. METHODS: Sputum from 50 patients with asthma was collected and processed using the standard processing protocol with one drop 20% albumin added to hinder cell smudging. Each slide was counted automatically using the CellaVision DM96 and manually by an experienced lab technician. Sputum was classified as eosinophilic or neutrophilic using 3% and 61% cutoffs, respectively. RESULTS: We found a good agreement using intraclass correlation for all target cells, despite significant differences in the cell count rate. The automated CDC had a sensitivity of 65%, a specificity of 93%, and a kappa-coefficient of 0.61 for identification of sputum eosinophilia. In contrast, the automated CDC had a sensitivity of 29%, a specificity of 100%, and a kappa-coefficient of 0.23 for identification of sputum neutrophilia. CONCLUSION: Automated- and manual cell counts of sputum agree with regards to the key inflammatory cells. The automated cell count had a modest sensitivity but a high specificity for the identification of both neutrophil and eosinophil asthma.

Differences between patients who achieved asthma control and those who remain uncontrolled after standardized severe asthma care strategy.

OBJECTIVE: To assess clinical, functional, and inflammatory patterns of children and adolescents with severe uncontrolled asthma, and investigate the differences between patients who achieved asthma control and those who remain uncontrolled after standardized asthma care strategy. METHODS: Screening all children and adolescents with asthma from the Pediatric Pulmonology Outpatient Clinic of Unicamp, Brazil, and included those with severe uncontrolled asthma according to GINA guidelines criteria. Patients were assessed at baseline and after by demographic and medication data, questionnaires (Asthma Control Test and Pediatric Asthma Quality of Life Questionnaire), Six-Minute Walk Test, skin prick test, spirometry, induced sputum, and blood collection (total immunoglobulin E and eosinophil count). Cytokine dosage was analyzed in sputum supernatant and serum by Cytometric Bead Array. RESULTS: Thirty-three patients with severe uncontrolled asthma were included (median age 10.9 [7.00-17.60] years). All patients presented satisfactory adherence to treatment and 50% of them achieved good asthma control after six-month follow-up (p < 0.001). Patients who achieved asthma control reported higher intervals since their last exacerbation episode (p = 0.008) and higher quality of life scores (p < 0.001) as compared to patients who remained uncontrolled. We found no changes in lung function markers, inflammatory biomarkers, or cytokine levels between patients with uncontrolled and controlled asthma. CONCLUSION: Participation of six months in a structured outpatient clinic for children with severe asthma had a notable improvement in control and quality of life of patients. This demonstrates the importance of a global assessment, focused on peculiarities presented by patients with severe uncontrolled asthma.

Sputum Inflammatory Patterns are Associated with Distinct Clinical Characteristics in Subjects with Occupational Asthma Independently from the Causal Agent.

BACKGROUND AND OBJECTIVES: Clinical heterogeneity in sensitizer-induced occupational asthma (OA) and its relationship to airway inflammatory profiles remain poorly elucidated. To further characterize the interactions between induced sputum inflammatory patterns, asthma-related outcomes and the high- or low-molecular-weight category of causal agents in a large cohort of subjects with OA. METHODS: This multicenter, retrospective, cross-sectional study was conducted among 296 subjects with OA ascertained by a positive specific inhalation challenge who completed induced sputum assessment before and 24 hours after challenge exposure. RESULTS: Multivariate logistic regression analysis revealed that sputum eosinophilia ≥3% was significantly associated with a high dose of inhaled corticosteroid (odds ratio [95% confidence interval], 1.31 [1.11-1.55] for each 250-µg increment in daily dose), short-acting b2-agonist use less than once a day (3.54 [1.82-7.00]), and the level of baseline nonspecific bronchial hyperresponsiveness (mild: 2.48 [1.21-5.08]); moderate/severe: 3.40 [1.44-8.29]). Sputum neutrophilia ≥76% was associated with age (1.06 [1.01-1.11]), male gender (3.34 [1.29-9.99]), absence of corticosteroid use (5.47 [2.09-15.16]), short-acting b2-agonist use once or more a day (4.09 [1.71-10.01]), ≥2 severe exacerbations during the last 12 months at work (4.22 [1.14-14.99]), and isolated early reactions during the SIC (4.45 [1.85-11.59]). CONCLUSIONS: The findings indicate that sputum inflammatory patterns in subjects with OA are associated with distinct phenotypic characteristics and further highlight the differential effects of neutrophils and eosinophils on asthma-related outcomes. These associations between inflammatory patterns and clinical characteristics share broad similarities with what has been reported in nonoccupational asthma and are not related to the type of causal agent.

Exploration of induced sputum BIRC3 levels and clinical implications in asthma.

BACKGROUND: Baculoviral IAP repeat-containing 3 (BIRC3) which encodes a member of the IAP family of proteins upregulated in the asthma expression profile dataset. However, there was few research on studying the clinical implication of BIRC3 in asthma. OBJECTIVE: To validate BIRC3 expression and its clinical implications in induced sputum of asthma. METHODS: Based on the GSE76262 (118 asthma cases and 21 healthy controls) dataset, differentially expressed genes were screened using R software. Subsequently, BIRC3 mRNA and protein were clinically verified in induced sputum samples through quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Besides, the correlations between BIRC3 expression and asthmatic eosinophilic/allergic inflammation indicators (FeNO, IgE, and EOS%), pulmonary function (FEV1, FEV1% pred, FVC% pred, and FEV1/FVC), and inflammatory cytokines (IL-4, IL-5, IL-13, IL-25, IL-10, IL-33, and TSLP) were analyzed. Finally, BIRC3 mRNA was detected in human primary bronchial epithelial cells stimulated by cytokines (IL-4 or IL-13). RESULTS: BIRC3 was screened as a candidate gene in the GSE76262, which was highly expressed in asthma. Highly expressed BIRC3 was positively correlated with eosinophilic and allergic indicators, including FeNO, blood eosinophil, and serum IgE. Moreover, BIRC3 protein was positively associated with inflammation cytokines, like IL-4, IL-5, IL-13, IL-25, IL-10, IL-33, and TSLP, while negatively correlated with FEV1, FEV1%pred, FVC% pred, and FEV1/FVC. Furthermore, the expression of BIRC3 could be induced in primary bronchial epithelial cells treated by cytokines IL-4 or IL-13. CONCLUSIONS: BIRC3 significantly increased in induced sputum of asthma and positively correlated with airway eosinophilic and peripheral blood allergic inflammation, type 2 cytokines, and airway obstruction. Increased BIRC3 might be involved in the pathogenesis of asthma by affecting the eosinophilic and allergic inflammation.

Induced sputum abnormalities in gas station attendants.

PURPOSE: To investigate inflammatory changes in the induced sputum (IS) of gas station attendants (GSAs) at risk of exposure to fuel vapors through inhalation and susceptible to respiratory complaints and impaired lung function. METHODS: Hypertonic saline-IS was collected from 52 GSAs who had never smoked (42 men, age = 35.9 ± 8.9 years) and had no known comorbidities. A group of 22 non-smokers (11 men, age = 30.5 ± 5.1 years) selected from the University staff served as control. The GSAs answered a questionnaire and underwent spirometry and chest tomography. A total of 15 inflammatory biomarkers associated with inflammation, including cytokines, chemokines, and mediators of immunological response, were also measured. RESULTS: The most common symptoms of GSAs were coughing (42.3%) and dyspnea (59.6%) based on the New York Heart Association (NYHA; Class II) classification. Significant elevations (p < 0.05) in forced vital capacity and forced expiratory volume within the first second were observed in the GSAs relative to those in the controls (GSA vs. control: 99 ± 12% vs. 90 ± 9% and 94 ± 11% vs. 87 ± 10%, respectively). The GSAs had a lower percentage of IS lymphocytes than that in the control group (4.5 ± 5.7% vs. 7.7 ± 9.8%). The GSAs also had significantly lower concentrations of IL-4, IL-5, IL-10, IL-12P70, IFN-γ, and MIP-1α, but IL-3 levels were higher. No differences were observed in the airway thickness and the amount of emphysema between the GSAs and the controls. CONCLUSION: Despite normal lung function and absence of abnormalities on HRCT, GSAs have a higher frequency of respiratory complaints, with evidence of impairment of lymphocytic activity in the airways.

No inflammatory effects after acute inhalation of barium sulfate particles in human volunteers.

BACKGROUND: Most threshold limit values are based on animal experiments. Often, the question remains whether these data reflect the situation in humans. As part of a series of investigations in our exposure lab, this study investigates whether the results on the inflammatory effects of particles that have been demonstrated in animal models can be confirmed in acute inhalation studies in humans. Such studies have not been conducted so far for barium sulfate particles (BaSO4), a substance with very low solubility and without known substance-specific toxicity. Previous inhalation studies with zinc oxide (ZnO), which has a substance-specific toxicity, have shown local and systemic inflammatory respones. The design of these human ZnO inhalation studies was adopted for BaSO4 to compare the effects of particles with known inflammatory activity and supposedly inert particles. For further comparison, in vitro investigations on inflammatory processes were carried out. METHODS: Sixteen healthy volunteers were exposed to filtered air and BaSO4 particles (4.0 mg/m3) for two hours including one hour of ergometric cycling at moderate workload. Effect parameters were clinical signs, body temperature, and inflammatory markers in blood and induced sputum. In addition, particle-induced in vitro-chemotaxis of BaSO4 was investigated with regard to mode of action and differences between in vivo and in vitro effects. RESULTS: No local or systemic clinical signs were observed after acute BaSO4 inhalation and, in contrast to our previous human exposure studies with ZnO, no elevated values of biomarkers of inflammation were measured after the challenge. The in vitro chemotaxis induced by BaSO4 particles was minimal and 15-fold lower compared to ZnO. CONCLUSION: The results of this study indicate that BaSO4 as a representative of granular biopersistent particles without specific toxicity does not induce inflammatory effects in humans after acute inhalation. Moreover, the in vitro data fit in with these in vivo results. Despite the careful and complex investigations, limitations must be admitted because the number of local effect parameters were limited and chronic toxicity could not be studied.

Etiology and the challenge of diagnostic testing of community-acquired pneumonia in children and adolescents.

BACKGROUND: Pneumonia is the leading cause of mortality in pediatric population. The etiology of pneumonia in this population is variable and changes according to age and disease severity and where the study is conducted. Our aim was to determine the etiology of community-acquired pneumonia (CAP) in children aged 1 month to 17 years admitted to 13 Colombian hospitals. METHODS: Prospective cohort study. Hospitalized children with radiologically confirmed CAP and ≤ 15 days of symptoms were included and followed together with a control group. Induced sputum (IS) was submitted for stains and cultures for pyogenic bacteria and Mycobacterium tuberculosis, and multiplex PCR (mPCR) for bacteria and viruses; urinary antigens for pneumococcus and Legionella pneumophila; nasopharyngeal swabs for viruses, and paired serology for atypical bacteria and viruses. Additional cultures were taken at the discretion of primary care pediatricians. RESULTS: Among 525 children with CAP, 71.6% had non-severe pneumonia; 24.8% severe and 3.6% very severe pneumonia, and no fatal cases. At least one microorganism was identified in 84% of children and 61% were of mixed etiology; 72% had at least one respiratory virus, 28% pyogenic bacteria and 21% atypical bacteria. Respiratory syncytial virus, Parainfluenza, Rhinovirus, Influenza, Mycoplasma pneumoniae, Adenovirus and Streptococcus pneumoniae were the most common etiologies of CAP. Respiratory syncytial virus was more frequent in children under 2 years and in severe pneumonia. Tuberculosis was diagnosed in 2.3% of children. IS was the most useful specimen to identify the etiology (33.6%), and blood cultures were positive in 3.6%. The concordance between all available diagnostic tests was low. A high percentage of healthy children were colonized by S. pneumoniae and Haemophilus influenzae, or were infected by Parainfluenza, Rhinovirus, Influenza and Adenovirus. CONCLUSIONS: Respiratory viruses are the most frequent etiology of CAP in children and adolescents, in particular in those under 5 years. This study shows the challenges in making an etiologic diagnosis of CAP in pediatric population because of the poor concordance between tests and the high percentage of multiple microorganisms in healthy children. IS is useful for CAP diagnosis in pediatric population.

Sputum microRNA-screening reveals Prostaglandin EP3 receptor as selective target in allergen-specific immunotherapy.

BACKGROUND: Several microRNAs (miRs) have been described as potential biomarkers in liquid biopsies and in the context of allergic asthma, while therapeutic effects on the airway expression of miRs remain elusive. In this study, we investigated epigenetic miR-associated mechanisms in the sputum of grass pollen-allergic patients with and without allergen-specific immunotherapy (AIT). METHODS: Induced sputum samples of healthy controls (HC), AIT-treated and -untreated grass pollen-allergic rhinitis patients with (AA) and without asthma (AR) were profiled using miR microarray and whole-transcriptome microarray analysis of the same samples. miR targets were predicted in silico and used to identify inverse regulation. Local PGE2  levels were measured using ELISA. RESULTS: Two hundred and fifty nine miRs were upregulated in the sputum of AA patients compared with HC, while only one was downregulated. The inverse picture was observed in induced sputum of AIT-treated patients: while 21 miRs were downregulated, only 4 miRs were upregulated in asthmatics upon AIT. Of these 4 miRs, miR-3935 stood out, as its predicted target PTGER3, the prostaglandin EP3 receptor, was downregulated in treated AA patients compared with untreated. The levels of its ligand PGE2 in the sputum supernatants of these samples were increased in allergic patients, especially asthmatics, and downregulated after AIT. Finally, local PGE2  levels correlated with ILC2 frequencies, secreted sputum IL-13 levels, inflammatory cell load, sputum eosinophils and symptom burden. CONCLUSIONS: While profiling the sputum of allergic patients for novel miR expression patterns, we uncovered an association between miR-3935 and its predicted target gene, the prostaglandin E3 receptor, which might mediate AIT effects through suppression of the PGE2 -PTGER3 axis.

Biomarkers for predicting response to aspirin therapy in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin desensitization followed by daily aspirin use is an effective treatment for aspirin-exacerbated respiratory disease (AERD). OBJECTIVE: To assess clinical features as well as genetic, immune, cytological and biochemical biomarkers that might predict a positive response to high-dose aspirin therapy in AERD. METHODS: We enrolled 34 AERD patients with severe asthma who underwent aspirin desensitization followed by 52-week aspirin treatment (650 mg/d). At baseline and at 52 weeks, clinical assessment was performed; phenotypes based on induced sputum cells were identified; eicosanoid, cytokine and chemokine levels in induced sputum supernatant were determined; and induced sputum expression of 94 genes was assessed. Responders to high-dose aspirin were defined as patients with improvement in 5-item Asthma Control Questionnaire score, 22-item Sino-Nasal Outcome Test (SNOT-22) score and forced expiratory volume in 1 second at 52 weeks. RESULTS: There were 28 responders (82%). Positive baseline predictors of response included female sex (p = .002), higher SNOT-22 score (p = .03), higher blood eosinophil count (p = .01), lower neutrophil percentage in induced sputum (p = .003), higher expression of the hydroxyprostaglandin dehydrogenase gene, HPGD (p = .004) and lower expression of the proteoglycan 2 gene, PRG2 (p = .01). The best prediction model included Asthma Control Test and SNOT-22 scores, blood eosinophils and total serum immunoglobulin E. Responders showed a marked decrease in sputum eosinophils but no changes in eicosanoid levels. CONCLUSIONS AND CLINICAL RELEVANCE: Female sex, high blood eosinophil count, low sputum neutrophil percentage, severe nasal symptoms, high HPGD expression and low PRG2 expression may predict a positive response to long-term high-dose aspirin therapy in patients with AERD.