Induced Sputum Levels Research Articles

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

BackgroundDue 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.MethodsAn 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.ResultsPLS-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.ConclusionsThis 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.Graphical

Sputum Biomarkers in Wood and Tobacco Smoke Etiotypes of Chronic Obstructive Pulmonary Disease.

There is a need to better understand the etiotypes of chronic obstructive pulmonary disease (COPD) beyond the tobacco-smoke (TS-COPD). Wood smoke COPD (WS-COPD) is characterized by greater airway compromise, milder emphysema, and slower rate of lung function decline than TS-COPD. However, it is unclear if these two etiotypes of COPD have differences in sputum biomarker concentrations. Objective was to compare sputum levels of selected sputum biomarkers between WS-COPD and TS-COPD, and healthy controls. Eighty-eight women (69±12 years) were recruited and classified into: WS-COPD (n=31), TS-COPD (n=29) and controls (n=28). Using ELISA, we determined induced sputum levels of metalloproteinase 9 (MMP-9), chemokine ligand 5 (CCL5), interleukin-8 (IL-8), chemokine ligand 16 (CCL16/HCC-4) and vascular endothelial growth factor (VEGF-1). Differences were analyzed by Kruskal-Wallis and Mann-Whitney-U tests and correlation between airflow limitation and biomarkers by Spearman's test. At similar degree of airflow obstruction, anthropometrics and medications use, the level of sputum CCL5 was higher in TS-COPD than WS-COPD (p=0.03) without differences in MMP-9, IL-8, CCL16/HCC-4, and VEGF-1. Women with WS-COPD and TS-COPD showed significantly higher sputum levels of MMP-9, IL-8 and CCL5 compared with controls (p<0.001). FEV1% predicted correlated negatively with levels of MMP-9 (rho:-0.26; P=0.016), CCL5 (rho:-0.37; P=0.001), IL-8 (rho:-0.42; P<0.001) and VEGF (rho:-0.22; P=0.04). While sputum concentrations of MMP-9, IL-8, and CCL5 were higher in COPD women compared with controls, women with TS-COPD had higher levels of CCL5 compared with those with WS-COPD. Whether this finding relates to differences in pathobiological pathways remains to be determined.

Pilot study of inflammatory biomarkers in matched induced sputum and bronchoalveolar lavage of 2-year-olds with cystic fibrosis.

In this pilot study, we investigated whether induced sputum (IS) could serve as a viable alternative to bronchoalveolar lavage (BAL) and yield robust inflammatory biomarkers in toddlers with cystic fibrosis (CF) featuring minimal structural lung disease. We collected IS, BAL (right middle lobe and lingula), and blood, and performed chest computed tomography (CT) scans from 2-year-olds with CF (N = 11), all within a single visit. Inflammatory biomarkers included 20 soluble immune mediators and neutrophil elastase (NE), as well as frequency and phenotype of T cells, monocytes/macrophages, and neutrophils. At the molecular level, nine mediators showed similar levels in IS and BAL (CXCL1, CXCL8, IL-1α, IL-1RA, IL-6, CCL2, CXCL10, M-CSF, VEGF-A), four were higher in IS than in BAL (CXCL5, IL-1β, CXCL11, TNFSF10), and two were present in IS, but undetectable in BAL (IL-10, IFN-γ). Meanwhile, soluble NE had lower activity in IS than in BAL. At the cellular level, T-cell frequency was lower in IS than in BAL. Monocytes/macrophages were dominant in IS and BAL with similar frequencies, but differing expression of CD16 (lower in IS), CD115, and surface-associated NE (higher in IS). Neutrophil frequency and phenotype did not differ between IS and BAL. Finally, neutrophil frequency in IS correlated positively with air trapping. IS collected from 2-year-olds with CF yields biomarkers of early airway inflammation with good agreement with BAL, notably with regard to molecular and cellular outcomes related to neutrophils and monocytes/macrophages.

Elevated levels of serum CDCP1 in individuals recovering from severe COVID-19 disease.

Background: COVID-19 survivors report residual lung abnormalities after discharge from the hospital. The aim of this study was to identify biomarkers in serum and induced sputum samples from patients after hospitalization for COVID-19.Methods: Patients admitted to hospitals in Spain with laboratory-confirmed COVID-19 were recruited for this study. SARS-CoV-2-infected patients were divided into groups with mild/moderate and severe disease according to the severity of their symptoms during hospitalization. Levels of 92 biomarkers were measured in serum and induced sputum samples.Results: A total of 108 patients (46.2% severe cases) were included in this study. The median number of days after the onset of symptoms was 104. A significant difference was observed in diffusing capacity for carbon monoxide (DLCO), an indicator of lung function, whereby DLCO <80% was significantly lower in severe cases (p <0.001). Differences in inflammatory biomarkers were observed between patients with mild/moderate and severe disease. For some biomarkers, correlations in serum and induced sputum levels were detected. Independent predictors of severe disease were DLCO <80% and the serum CDCP1 value.Conclusions: Higher levels of CDCP1 remain after hospital discharge and are associated with the severity of COVID-19. The possible prognostic implications warrant further investigation.

Inflammatory profile of induced sputum composition in systemic sclerosis and comparison with healthy volunteers

Systemic sclerosis (SSc) is a potentially serious and disabling connective tissue disease specially in case of interstitial lung disease (SSc-ILD). The aim of our study was to evaluate the potential utility of dosing in the induced sputum (IS) and to compare their levels in SSc-ILD and SSc-nonILD patients, as well as in healthy volunteers (HV). IS and sera values were also compared. In a prospective cross-sectional analysis, we studied the IS and serum provided from 25 SSc patients, 15 SSc-nonILD and 10 SSc-ILD, compared to 25 HV. We analyzed sputum cell composition and quantified in the supernatant and corresponding serum by commercially available immunoassays: IGFBP-1, IGFBP-2, IGFBP-3, TGF-β, IL-8, TNF-α, YKL-40, MMP-7 and MMP-9. Lung function was studied by the determination of FEV-1 (%), FVC (%), DLCO (%) and KCO (%). The IS of SSc patients had a lower weight than HV (p<0.05, p<0.01) without any significant difference with regard to the cellularity. IGFBP-1 (p < 0.0001), TGF-β (p < 0.05), IL-8 (p < 0.05), YKL-40 (p < 0.0001) and MMP-7 (p < 0.01) levels were increased in the IS of SSc patients compared to HV. Only IL-8 serum levels (p < 0.001) were increased in SSc patients compared to HV. Neither in IS nor in serum were observed differences between SSc-ILD and SSc-nonILD patients. Correlations were observed between IS IL-8 levels and FEV-1 (%) (r = = − 0.53, p < 0.01), FVC (%) (r = − 0.51, p < 0.01) and annualized ∆KCO (%) (r = 0.57, p < 0.05), between IS TGF-β levels and annualized ∆FEV-1 (%) (r = = − 0.57, p < 0.05), between IS IGFBP-2 levels and annualized ∆KCO (%) (r = 0.56, p < 0.05). Our study showed that SSc patients exhibit raised IS levels of IGFBP-1, TGF-β, IL-8, YKL-40 and MMP-7, molecules known to be involved in lung remodeling and fibrotic process, without any significant difference between SSc-ILD and SSc-nonILD patients. IL-8, TGF-β and IGFBP-2 are correlated with lung function in SSc patients which emphasize clinical relevance. IS analysis represents a new approach to understand lung inflammatory process in SSc patients. A longitudinal study is needed to evaluate their pathophysiological relevance.

Periostin and Thymic Stromal Lymphopoietin-Potential Crosstalk in Obstructive Airway Diseases.

Periostin and thymic stromal lymphopoietin (TSLP) are newly described markers of obstructive airway diseases and the mechanism by which both markers participate in immune response remains poorly understood. The aim of our study was to determine periostin and TSLP concentration in serum and induced sputum (IS) in patients with atopic asthma, chronic obstructive pulmonary disease (COPD), and controls, as well as to evaluate the potential link between periostin, TSLP, and Th2 immune response. Serum and IS levels of periostin, TSLP, IL-4, and IL-13 were determined in 12 atopic asthmatics, 16 COPD sufferers, and 10 controls. We noticed a significantly higher IS periostin and TSLP concentration at protein and mRNA level in asthmatics compared to the two other groups; additionally, periostin and TSLP were correlated positively with IS eosinophil count. A strong positive correlation between IS periostin and TSLP protein levels (r = 0.96) as well as mRNA expression level (r = 0.95) was found in patients with asthma. The results of our study show that periostin and TSLP are associated with eosinophilic airway inflammation and seem to be important drivers of atopic asthma but not COPD pathobiology. Very strong correlations between local periostin, TSLP, eosinophils, and IL-4 in asthma point to the link between periostin–TSLP and Th2 response.

Pulmonary functions in children and adolescents with sickle cell disease.

Sickle cell disease (SCD) is relatively common in Bahrain, and airway inflammation in patients with SCD is usually multifactorial. This study aimed to evaluate lung function and induced sputum levels of interleukin-6 (IL-6) in Bahraini children and adolescents with SCD and assess their relationship with the recurrence of acute chest syndrome (ACS). A total of 139 children and adolescents with SCD and 123 healthy children (control group) were included in the present study. Patients were further stratified according to age and history of ACS. The patient and control groups underwent pulmonary function tests (PFTs), including spirometry and assessments of lung volume, diffusion of carbon monoxide (DLCO), and induced sputum IL-6 levels. Forced expiratory volume in 1 second (FEV1 ), force vital capacity (FVC), FEV1 /FVC, total lung capacity, DLCO, and DLCOc (ie, hemoglobin-corrected DLCO) were significantly lower, while residual volume and sputum IL-6 levels were significantly higher in the patient group than in the control group. PFT parameters were more compromised in the patient subgroup with a history of ACS and older than 12 years compared with the subgroup without a history of ACS and the subgroup under 12 years of age. PFTs revealed significant negative correlations with age, number of ACS events, and sputum IL-6 levels. Pulmonary function was observed to worsen with disease progression, and it worsened with older age and repeated occurrence of ACS. Induced sputum IL-6 levels reflected the degree of lung inflammation in affected patients and were associated with more impairment in various PFT parameters.

Динамика лабораторных показателей у детей с легкой персистирующей бронхиальной астмой

Study Objective: to track the laboratory dynamics in children with mild persistent bronchial asthma (BA) at various stages of disease with or without baseline therapy. Study Design: prospective cohort study. Materials and Methods. The study included 42 patients with mild persistent BA aged 5 to 16 years. Each child was followed up for 3 years. At initiation of the study and during each visit, all patients had their blood eosinophils, nasal discharge (ND) and induced sputum (IS), as well as serum eosinophil cation protein (ECP) tested; and their respiratory function was assessed. All children were prescribed baseline therapy with low doses of inhalative glucocorticosteroids (GCS); 18 children also received allergen-specific immunotherapy (ASIT). Study Results. Inhalative GCS for BA exacerbations facilitated normalisation of sputum, ND and blood eosinophils. During periods of BA remission, ND and IS eosinophil levels increased, but patients did not have rhinitis and BA symptoms. Eosinophil levels in IS were higher vs. BA control (р = 0.05) and lower vs. disease exacerbations (р &lt; 0.05). We did not find any differences in laboratory results in children with or without ASIT who were treated with low doses of inhalative and nasal GCS. Conclusion. Eosinophile inflammatory phenotype in patients did not charge depending on disease duration and control therapies. According to the study data, eosinophile levels in IS, when BA exacerbation was recorded, were 8.0% [3.0–16.0%]. This value can be used as an additional predictor for the need in increased therapy (a step forward). Irrespective of the disease stage and therapy, there was correlation between blood eosinophile level and IS eosinophile level and between IS eosinophile level and blood ECP concentration. Keywords: bronchial asthma, children, eosinophiles, induced sputum, inflammatory phenotype.

Characterization of sputum biomarkers for asthma-COPD overlap syndrome.

Asthma–COPD overlap syndrome (ACOS) is a commonly encountered chronic airway disease. However, ACOS is still a consensus-based clinical phenotype and the underlying inflammatory mechanisms are inadequately characterized. To clarify the inflammatory mediatypical for ACOS, five biomarkers, namely interleukin (IL)-13, myeloperoxidase (MPO), neutrophil gelatinase-associated lipocalin (NGAL), chitinase-like protein (YKL-40), and IL-6, were selected. This study hypothesized that sputum biomarkers relevant for airway inflammation in asthma (IL-13), COPD (MPO, NGAL), or in both asthma and COPD (YKL-40, IL-6) could be used to differentiate ACOS from COPD and asthma. The aim of this study was to characterize the inflammatory profile and improve the recognition of ACOS. Induced sputum levels of IL-13, MPO, NGAL, YKL-40, and IL-6 were measured by enzyme-linked immunosorbent assay/Luminex assay in a Finnish discovery cohort (n=90) of nonsmokers, smokers, and patients with asthma, COPD, and ACOS and validated in a Japanese cohort (n=135). The classification accuracy of potential biomarkers was compared with area under the receiver operating characteristic curves. Only sputum NGAL levels could differentiate ACOS from asthma (P<0.001 and P<0.001) and COPD (P<0.05 and P=0.002) in the discovery and replication cohorts, respectively. Sputum NGAL levels were independently correlated with the percentage of pre-bronchodilator forced expiratory volume in 1 second predicted in multivariate analysis in the discovery and replication cohorts (P=0.001 and P=0.002, respectively). In conclusion, sputum biomarkers reflecting both airway inflammation and remodeling of the tissue show potential in differentiation between asthma, COPD, and ACOS.

Biological monitoring of particulate matter accumulated in the lungs of urban asthmatic children in the Tel-Aviv area.

Lung inflammation from exposure to airborne particulate matter (PM) may be responsible for morbidity in asthma, but several studies using environmental monitoring data showed inconsistent results. Thus, the aim of this study was to evaluate the capability of induced sputum (IS) technology in order to biologically monitor PM in the lungs of urban asthmatic children. We collected clinical, demographic, biological and environmental monitoring data on 136 children referred for asthma evaluations. The study participants were divided into two groups according to IS eosinophil counts of <3% (non-eosinophilic inflammation, n = 52) and ≥3% (eosinophilic inflammation, n = 84). The eosinophilic group displays significantly higher levels of fractional exhaled nitric oxide than the non-eosinophilic one (58.8 ± 47.5 vs 28.9 ± 34.2 ppm, p = 0.007). Particles (0-2.5 and 0-5 µm) comprised a strong risk factor for eosinophilic inflammation in IS (≥3%). Children with >80% of particles (0-2.5 µm) out of the total PM accumulated in the airways displayed the highest OR 10.7 (CI 2.052-56.4 p = 0.005) for an existing eosinophilic inflammation. Heme oxygenase-1 (HO-1) enzyme levels in IS positively correlated with % eosinophils and with particles in IS ranging between 2 and 3 μm. The level of HO-1 enzyme activity and FEV1/FVC in children with <3% eosinophils, but not ≥3%, was positively and significantly correlated, showing a protective effect of HO-1. Accumulation of PM involves oxidative stress pathways and is a risk factor for developing eosinophilic inflammation in asthmatic children. IS can biologically monitor this process.

Induced sputum levels of IL-33 and soluble ST2 in young asthmatic children

Objective: Interleukin-33 is an IL-1 family cytokine which signals via its T1/ST2 receptor, and acts as a key regulator of inflammation, notably the type-2 response implicated in asthma. This study aims to measure the expression of soluble ST2 (sST2) and IL-33 in asthmatic children, depending on disease activity. Methods: Thirty-seven children with well-defined asthma (20 moderate and 17 mild asthmatics) were studied. IL-33 and sST2 were measured by ELISA in serum and induced sputum (IS) samples, and compared with 22 age- and sex-matched healthy controls. Real-time quantitative PCR was used to determine IL-33 and TNF-α mRNA expression in IS. Results: sST2 and IL-33 levels in IS and serum were significantly higher in patients compared with healthy controls (p = 0.0001). The increase in sST2 and IL33 was significantly more important in moderate cases than in mild asthma. A significant correlation was observed between serum and IS IL-33 levels (r = 0.497; p = 0.0018). Higher levels of IL-33 mRNA were detected in IS from asthmatics than those observed in controls. A significant correlation was found between TNF-α and IL-33 mRNA expression in the asthmatic subjects (r = 0.772, p = 0.0001). Conclusions: Values of sST2 and IL-33 observed in IS were found to correlate with disease activity. Elevated IL-33 mRNA expression in IS and its correlation with TNF-α reflected the inflammatory process observed in the lung of young asthmatics.

Differences in plasma and sputum biomarkers between COPD and COPD–asthma overlap

The pathophysiological features of chronic obstructive pulmonary disease (COPD)-asthma overlap are poorly understood and there has been no study of plasma or sputum biomarkers in overlap patients. In order to clarify the similarity and differences between overlap and COPD or asthma, we have investigated four potential biomarkers of COPD: surfactant protein A (SP-A), soluble receptor for advanced glycation end-products (sRAGE), myeloperoxidase (MPO) and neutrophil gelatinase-associated lipocalin (NGAL). SP-A and sRAGE are pneumocyte-derived markers. MPO and NGAL are neutrophil-derived molecules, but NGAL can also be expressed by respiratory epithelial cells. Plasma levels of SP-A and sRAGE and induced sputum levels of MPO and NGAL were measured by enzyme immunoassay/ELISA in 134 subjects: nonsmokers (n=26), smokers (n=23), asthma (n=32), COPD (n=39) and COPD-asthma overlap patients (n=14). In patients with COPD-asthma overlap, sputum MPO and plasma SP-A were significantly elevated whereas plasma sRAGE levels were reduced compared with asthma patients. Only sputum NGAL was significantly elevated in COPD-asthma overlap compared with COPD (p=0.00016) and could be used to differentiate patients with overlap from those with COPD. Increased induced sputum levels of NGAL might be a characteristic feature of overlap, suggesting enhanced neutrophilic airway inflammation and/or airway epithelial injury in COPD-asthma overlap.

The effect of human antibacterial peptide LL-37 in the pathogenesis of chronic obstructive pulmonary disease

Previous research has shown that innate immune system was more important than the acquired immune system in the pathogenesis of COPD. LL-37 is the only human cathelicidin identified so far. As an integral part of the innate immune system, besides antibacterial activity, its chemotactic activity, damage repairing, influencing apoptosis and its cytotoxicity are attracting people's attention. The aim of the present study was to evaluate role of LL-37 in the pathogenesis of COPD. ELISA and immunohistochemistry were applied to investigate the expression of LL-37 in induced sputum and lung tissue of COPD patients. Bronchial epithelial cell (BEP2D) and alveolar epithelial cell (A549) were treated with LL-37 synthesis polypeptide invitro to assess the role of LL-37 in inflammation and apoptosis. We found that increased induced sputum levels of LL-37 in COPD patients were associated with airflow limitation, health status and exercise tolerance and the expressing intensity of LL-37 in both airway district and pulmonary alveoli area in COPD group significantly increased compared with control group. Through stimulation by CSE and LPS, the expression of LL-37 was increased in bronchial epithelial cell and alveolar epithelial cell. LL-37 synthesis polypeptide can promote the releasing of inflammatory factor IL-8 and induce apoptosis of bronchial epithelial cell and alveolar epithelial cell. This study suggested that LL-37 may play important role in the pathogenesis of COPD and may be a possible novel therapeutic target in COPD.

Urokinase‐type plasminogen activator system and human cationic antimicrobial protein 18 in serum and induced sputum of patients with chronic obstructive pulmonary disease

There is increasing evidence that the innate immune system plays an important role in the pathogenesis of COPD. The objective of this study was to quantify several innate immune biomarkers in serum and induced sputum of COPD patients, and healthy non-smokers and smokers. Serum and induced sputum levels of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator inhibitor (PAI-1) and human cationic antimicrobial protein 18 (CAP18) were measured by ELISA, in 13 patients with stage I or stage II COPD (COPD I + II), 15 patients with stage III or stage IV COPD (COPD III + IV), 18 healthy non-smokers and 14 healthy smokers. In addition, membrane-bound uPAR in peripheral blood and induced sputum was assessed by flow cytometry. Levels of uPAR, PAI-1 and CAP18 were elevated in induced sputum of COPD I + II and COPD III + IV patients, compared with healthy non-smokers (P < 0.05) and healthy smokers (P < 0.05). uPAR, PAI-1 and CAP18 levels were significantly higher in COPD III + IV patients compared with COPD I + II patients (P < 0.05). The expression of uPAR on induced sputum neutrophils and macrophages was significantly higher in COPD patients compared with healthy non-smokers (P < 0.05) and healthy smokers (P < 0.05). Sputum uPAR and CAP18 levels showed significant inverse correlations with FEV(1)% and 6MWD, and significant positive correlations with St. George's Respiratory Questionnaire scores. In COPD patients, increased induced sputum levels of uPAR, PAI-1 and CAP18 were associated with airflow limitation, health status and exercise tolerance, suggesting that these biomarkers may be implicated in the pathogenesis of COPD.

PLASMINOGEN ACTIVATOR INHIBITOR-1 PROMOTES INFLAMMATORY PROCESS INDUCED BY CIGARETTE SMOKE EXTRACTION OR LIPOPOLYSACCHARIDES IN ALVEOLAR EPITHELIAL CELLS

Plasminogen activator inhibitor-1 (PAI-1) plays a role in regulating levels of some cytokines and cell migration in addition to its classic role in inhibiting fibrinolysis. PAI-1 levels in induced-sputum of chronic obstructive pulmonary disease (COPD) patients were elevated significantly and correlated negatively with pulmonary function. To study the role of PAI-1 in inflammatory process in COPD, the authors transfected alveolar epithelial cells (AECs) with siRNA-targeted PAI-1 and stimulated the cells by cigarette smoke extraction (CSE) or lipopolysaccharides (LPS). The expression of inflammatory factors, interleukin-8 (IL-8) and leukotriene B4 (LTB4), and the monocyte migration were detected. The exposure to CSE or LPS induced the expression of PAI-1, IL-8, and LTB4 in AECs and monocyte migration to AECs. However, they were attenuated after transfection with siRNA-targeted PAI-1. In conclusion, PAI-1 stimulates inflammation induced by CSE or LPS in AECs through up-regulation of inflammatory factors and promoting inflammatory cell migration. PAI-1 may play a proinflammatory role in the pathogenesis of COPD.

Induced sputum matrix metalloproteinase‐9 correlates with lung function and airway inflammation in children with cystic fibrosis

Matrix metalloproteinases (MMPs) degrade extracellular matrix and are implicated in causing airway damage in chronic inflammatory lung diseases, including cystic fibrosis (CF). Our primary objective was to examine the relationship between matrix metalloproteinase-9 (MMP-9) and pulmonary function, as measured by forced expiratory volume in 1 sec (FEV1), in children with CF. We measured MMP-9 and its natural tissue inhibitor of metalloproteinase-1 (TIMP-1) in induced sputum from 18 clinically stable CF children with normal to mildly abnormal lung function and 7 healthy control children. Measures of airway inflammation from induced sputum included cell counts and differentials, interleukin-8 (IL-8), neutrophil elastase, MMP-9, and TIMP-1. Infection was assessed through quantitative bacterial counts. Induced sputum levels of MMP-9 and TIMP-1 were significantly increased in children with CF compared with healthy controls. Also, the MMP-9/TIMP-1 molar ratio was higher in the CF group. Among CF children, there was a significant inverse relationship between MMP-9 and FEV1. In addition, sputum MMP-9 and TIMP-1 concentrations significantly correlated with total white cells and neutrophils, IL-8, and neutrophil elastase. Neither MMP-9 nor TIMP-1 correlated with airway infection. We conclude that clinically stable CF children with normal to mildly abnormal lung function have an increased burden of MMP-9 in their airways. The observed relationships of MMP-9 with lung function and other measures of airway inflammation suggest that this enzyme may be a useful marker of airway injury and airflow obstruction in persons with CF.

Comparison between exhaled and sputum oxidative stress biomarkers in chronic airway inflammation.

The aim of the present study was to compare aldehyde levels resulting from lipid peroxidation in exhaled breath condensate (EBC) and induced sputum (IS) supernatant of subjects with asthma and chronic obstructive pulmonary disease (COPD). Aldehydes (malondialdehyde (MDA), acrolein, n-hexanal (C6), n-heptanal (C7), n-nonanal (C9), 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE)) in both biological fluids were measured by liquid chromatography-tandem mass spectrometry. MDA concentrations in sputum were 132.5 nM (82.5-268.8) and 23.7 nM (9-53.7) in EBC. Similarly, C6, C7 and C9 concentrations in IS were 1.5-4.7-fold higher than in EBC. Acrolein levels were 131.1 nM (55.6-264.6) in IS and 45.3 nM (14.4-127.1) in EBC. The concentrations of HNE and HHE in IS were not significantly different from the levels in EBC. Aldehyde levels in EBC did not show any correlation with aldehyde levels in IS or with differential sputum cellular count. In COPD, MDA in EBC, but not its IS counterpart, was negatively correlated with the severity of disease. In conclusion, the data presented here show that aldehydes can be detected in both exhaled breath condensate and supernatant of induced sputum, but that their relative concentrations are different and not correlated with each other. Therefore, with regard to lipid peroxidation products, exhaled breath condensate and induced sputum must be considered as independent techniques.

Safety and use of sputum induction in children with cystic fibrosis

We assessed the safety and use of induced sputum (IS) in children with cystic fibrosis (CF). Forty-eight children (19 males) with CF, mean age 12.6 (range, 7.3-17.0) years and median forced expired volume in 1 sec (FEV(1)) 48% (range, 14-77%) predicted were recruited. Patients spontaneously expectorated sputum and then performed sputum induction by inhalation of nebulized 7% hypertonic saline. Samples were sent for bacteriological culture, and for measurement of the following inflammatory mediators: interleukin-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase activity. FEV(1) was performed before and after inhalation of hypertonic saline. There was no increase in mediator levels in IS compared to expectorated sputum (ES) samples. Only 3 patients demonstrated significant bronchoconstriction following inhalation of hypertonic saline, by the method used. From the ES samples, Pseudomonas aeruginosa was isolated in 13 patients, Staphylococcus aureus in 7 patients, Stenotrophomonas maltophilia in 1 patient, and both Pseudomonas aeruginosa and Staphylococcus aureus in 5 patients. All these organisms were found in the IS samples. However, in 2 patients whose ES grew no organisms, one patient's IS grew Pseudomonas aeruginosa, and the other patient's IS grew Staphylococcus aureus. In our study, sputum induction was safe, with no proinflammatory effect.

Induced sputum-retrieved matrix metalloproteinase 9 and tissue metalloproteinase inhibitor 1 in granulomatous diseases.

Matrix metalloproteinases (MMPs) capable of degrading various components of connective tissue matrices, and tissue inhibitor metalloproteinases (TIMPs) are considered important in lung parenchymal remodeling and repair processes in pulmonary diseases. Induced sputum (IS) is a reliable noninvasive method to investigate pathogenesis, pathophysiology and treatment of lung disease. This study was designed to determine whether IS-MMP9/TIMP1 levels demonstrate lung parenchymal remodeling in sarcoidosis (SA) and Crohn's disease (CRD) patients. Sputum was induced and processed conventionally in 13 SA patients, 18 CRD patients and 9 controls. Two-hundred cells were counted on Giemsa-stained cytopreps, and T lymphocytes subsets (CD4 = T helper and CD8 = T suppressor cytotoxic cells) were analysed by FACS using monoclonal antibodies.MMP-9 and TIMP-1 were measured using commercial ELISA kits. MMP-9 concentrations, but not those of TIMP-1, were significantly greater in the sputum supernatant in SA and CRD patients compared to controls (P = 0.018 and P = 0.0019, respectively). The molar ratio, MMP-9/TIMP-1, was significantly higher in SA and CRD patients compared to controls (P = 0.008 and P = 0.024, respectively). Gelatinase species having a molecular weight similar to that of MMP-9 were demonstrated by zymographic analysis. MMP-9 levels were highly correlated with the CD4/CD8 ratio and DLCO capacity in SA but less in CRD patients. MMP-9 levels in IS provide a sensitive marker for pulmonary damage.

COMPARISON OF INFLAMMATORY ELEMENTS IN NASAL LAVAGE AND INDUCED SPUTUM FOLLOWING OCCUPATIONAL EXPOSURE TO MOLDY-BUILDING MICROBES

Inflammatory processes in the nasal air passages may reflect corresponding processes in the lower airways due to the similarities in histology of nasal mucosa and bronchi. The objective of the current study was to determine whether the levels of inflammatory markers in nasal lavage fluid could be used as predictors of lower respiratory tract inflammation after exposure to microbes in indoor air of moisture-damaged buildings. Differential cell count, immunochemically measured concentrations of proinflammatory cytokines (Interleukins [IL] IL-1, IL-4, IL-6, and tumor necrosis factorα [TNFα]) and nitric oxide (NO), assessed as nitrite, were analyzed from nasal lavage (NL) and induced sputum (IS) samples of the occupants (n = 60) working in moisture-damaged and reference school buildings. The measurements of inflammation markers in NL and IS sample pairs, collected on the same day, were compared. Although the levels of NO (p = .026) and IL-4 (p = .014) in NL predicted their levels in IS in a statistically significant manner, their predictive values (6.9% and 7.8%, respectively) were low. There was no significant correlation between the concentrations of the studied proinflammatory cytokines or differential cell counts in NL and IS samples. Our results indicate that measurement of inflammatory mediators in NL is not per se a reliable method to evaluate the inflammatory status of the lower airways after exposure to indoor air pollutants of moisture damaged building. It is possible that NL is a more sensitive indicator of direct exposure to different irritants in inhaled air than is IS. This may be a reflection of the role of nasal mucosa as the primary physicochemical barrier to inhaled air.