Increased inflammatory markers in the exhaled breath condensate of cigarette smokers.

Cigarette smoking induces an inflammatory response in the airways that may play a key role in the pathogenesis of chronic obstructive pulmonary disease. Noninvasive markers of inflammation may, therefore, be useful in monitoring the airways of smokers as well as in the screening of subjects at high risk of developing airway obstruction. The aim of the present study was to determine whether the concentrations of the pro-inflammatory cytokine, interleukin (IL)-6, is increased in the exhaled breath condensate of smokers and whether the number of cigarettes smoked has any influence on the exhaled concentrations. The possibility that exhaled IL-6 levels are related to exhaled carbon monoxide (CO) and lung function has also been explored. Another inflammatory marker, leukotriene (LT), was also measured. Twenty-one smokers (39+/-7 yrs, 13 male) and 14 nonsmokers (45+/-6 yrs, eight male) were recruited. IL-6 and LTB4 levels in the breath condensate were measured with an immunoassay kit and exhaled CO examined by means of a modified electrochemical sensor. Higher IL-6 and exhaled CO concentrations were found in current smokers (5.6+/-1.4 pg x mL(-1) and 16.7+/-5.5 parts per million (ppm)) than in nonsmokers (2.6+/-0.2 pg x mL(-1) and 2.1+/-0.6 ppm). Elevated concentrations of LTB4 were also observed in smokers compared to nonsmokers (9.4+/-0.4 pg x mL(-1) versus 6.1+/-0.3 pg x mL(-1)). In addition, there was a correlation between IL-6 concentrations, the number of cigarettes smoked per day, exhaled CO, LTB4 and lung function. Exhaled interleukin-6 and leukotriene B4 levels may be useful noninvasive markers of airway inflammation in cigarette smokers.     

Increased interleukin-4 and decreased interferon-gamma in exhaled breath condensate of children with asthma

Exhaled breath condensate analysis for noninvasive quantification of airway inflammation in asthma is a potentially useful research tool in children. There is an imbalance between T-helper (Th)-2 cells, which secrete interleukin (IL)-4, and Th1 cells, which secrete interferon (IFN)-gamma, in asthma. We measured concentrations of IL-4 and IFN-gamma in breath condensates of 37 children (11 normal, 12 steroid-naive, and 14 steroid-treated children with asthma). Exhaled IFN-gamma was significantly lower in steroid-naive and steroid-treated children with asthma compared with normal control subjects (3.7 +/- 0.2 versus 5.1 +/- 0.4 pg/ml, p < 0.01 and 4.1 versus 5.1 pg/ml, p < 0.05). By contrast, mean exhaled IL-4 was elevated in asthma (53.7 +/- 4.2 pg/ml) compared with normal children (35.7 +/- 6.2 pg/ml, p < 0.05) and concentrations were lower with steroid treatment (37.5 +/- 5.6 pg/ml, p < 0.05). Exhaled IL-4 was significantly lower in children with asthma on more than 600 microg inhaled steroid/day. The IL-4/IFN-gamma ratio was significantly greater in children with asthma compared with control children and the children with asthma on inhaled steroid therapy. We have shown for the first time that IFN-gamma and IL-4 can be assayed in exhaled breath condensate and shows an increased ratio of IL-4/IFN-gamma, consistent with predominance of Th2 cells in airways of children with asthma. Exhaled breath condensate analysis may have a useful role in studying allergic inflammation in childhood asthma.     

High levels of interleukin-6 in the exhaled breath condensate of patients with COPD

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by chronic inflammation of the respiratory tract. METHODS: We investigated the presence of interleukin-6 (IL-6: a cytokine secreted by monocytes/macrophages, T cells, B cells, fibroblasts, bone marrow stromal cells, keratinocytes and endothelial cells) in the exhaled breath condensate of 16 exsmokers with moderate COPD, 12 healthy non-smokers. IL-6 was measured by means of a specific enzyme immunoassay. RESULTS: IL-6 levels were detectable in all of the subjects, but were higher in the COPD patients (8.0 +/- 0.1 pg/ml; P < 0.0001) than in the healthy non-smokers (4.9 +/- 0.2 pg/ml) with a correlation in this group between age and IL-6 levels (r = 0.597; P < 0.05). CONCLUSIONS: The increased IL-6 levels in exhaled breath condensate may reflect airway inflammation in patients with COPD.     

Increased leukotrienes in exhaled breath condensate in childhood asthma

Cysteinyl leukotrienes (cys-LTs; LTC4, LTD4, and LTE4) are generated predominantly by mast cells and eosinophils and induce airway smooth muscle contraction, microvascular leakage, and mucous hypersecretion whereas leukotriene B4 (LTB4) is a potent chemoattractant of neutrophils. We measured cys-LTs and LTB4 in exhaled breath condensate from children aged 7-14 years including healthy nonatopic children (n = 11) and children with mild intermittent asthma (steroid naive, n = 11), mild persistent asthma (low-dose inhaled steroid treatment, n = 13), or moderate to severe persistent asthma (high-dose inhaled steroid treatment, n = 13). Exhaled LTB4 levels were increased in patients with mild and moderate to severe persistent asthma compared with patients with mild intermittent asthma (126.0 +/- 8.8 and 131.9 +/- 7.1 versus 52.7 +/- 3.8 pg/ml, p < 0.001 and p < 0.0001) and normal subjects (126.0 +/- 8.8 and 131.9 +/- 7.1 versus 47.9 +/- 4.1 pg/ml, p < 0.0001). Elevated exhaled cys-LT levels were found in patients with mild and moderate to severe persistent asthma compared with normal subjects (27.9 +/- 2.8 and 31.5 +/- 4.5 versus 18.5 +/- 0.5 pg/ml, p < 0.01 and p < 0.05). There was an inverse correlation between exhaled cys-LTs and LTB4 in patients with mild persistent asthma. We conclude that exhaled cys-LTs and LTB4 may be noninvasive markers of airway inflammation in pediatric asthma.     

Inflammatory response to sputum induction measured by exhaled markers

BACKGROUND: Sputum induction is increasingly used to study both cellular and biochemical composition of the airways. However, there is a significant rise in the percentage of neutrophils at 8 h after inhalation with hypertonic saline. OBJECTIVE: The aim of this study was to assess whether markers of inflammation in exhaled air and exhaled air condensate change after sputum induction in normal and asthmatic subjects. METHODS: We measured leukotriene B(4) (LTB(4)) and a marker of oxidative stress, 8-isoprostane, (by enzyme immunoassay) in exhaled air condensate and exhaled nitric oxide (NO; by chemiluminescence analyzer) in 15 healthy subjects (8 females, mean age 35 +/- 4 years, FEV(1) 97.4% predicted) and in 8 mild asthmatic subjects (5 males, mean age 34 +/- 2 years, FEV(1) 70.5% predicted). RESULTS: LTB(4) was significantly higher compared with baseline at 6 h but did not remain increased at 24 h after sputum induction (134.3 +/- 30.15 and 75.4 +/- 14.32 vs. 64.6 +/- 11.6 pg/ml at baseline; p < 0.02 and p > 0.05, respectively) in healthy subjects. An inverse correlation between LTB(4) and exhaled NO at 6 h after sputum induction was observed in healthy subjects (r = -0.66, p < 0.03). No increase in LTB(4) levels was observed in asthmatic patients. Baseline 8-isoprostane levels were higher in asthmatic patients than in healthy subjects (47.3 +/- 37.1 vs. 17.5 +/- 8.8 pg/ml; p < 0.01). A trend towards increased levels of 8-isoprostane could be observed at 6 and 24 h after inhalation in healthy subjects (26.2 +/- 3.7 and 26.7 +/- 3.9 pg/ml; p = 0.09 and p = 0.07, respectively). In healthy subjects, exhaled NO was significantly higher compared with baseline at 6 h and remained increased 24 h after sputum induction (7.96 +/- 3.5 vs. 5.61 +/- 1.86 ppb; p < 0.01 and p < 0.05, respectively). Exhaled NO levels were increased in asthmatic patients but did not further increase after sputum induction. CONCLUSIONS: Sputum induction with hypertonic saline causes an inflammatory response which should be considered when using the technique to monitor airway inflammation.     

Adenosine 5'-monophosphate increases levels of leukotrienes in breath condensate in asthma

Hyperresponsiveness (AHR) is a key physiological abnormality in asthma. In clinical and research studies AHR is measured bronchial challenge, with methacholine (MCh), but more recently with adenosine-5'-monophosphate (AMP). In the search for markers of airway inflammation in asthmatic patients, we measured the concentrations of histamine and cysteinyl-leukotrienes (cys-LTs) before and after MCh and AMP challenges in the exhaled breath condensate of 13 patients with mild asthma (FEV1 78.5%pred) and nine healthy non-smokers, using specific enzyme immunoassays. With methacholine challenge we did not find any differences between asthmatics and normal subjects in the pre- and post-challenge concentrations of cys-LTs: 27.2+/-1.4 vs. 29.2+/-1.2 pg/ml and 26.3+/-2.2 vs. 27.5+/-4.2 pg/ml, respectively or histamine: 5.1+/-0.4 vs. 5.1+/-0.6 nM and 4.5+/-0.4 vs. 4.4+/-0.3 nM; P>0.05). In asthmatic patients cys-LT levels were significantly higher after AMP challenge (56.2+/-9.7 vs. 31.7+/-6.9 pg/ml; P<0.05); but there was no difference in healthy subjects (27.2+/-4.6 vs. 30.3+/-4.7 pg/ml). There was no difference in histamine concentrations in asthmatic (5.9+/-1.8 vs. 4.5+/-0.5 nM), or healthy subjects (5.5+/-0.4 vs.5.7+/-0.9 nM) after AMP challenge. In conclusion, our results show that the cys-LTs are increased in exhaled breath condensate after AMP challenge, which may indicate that the AMP acts indirectly by releasing cys-LTs from primed mast cells. The detection of LTs and histamine in exhaled breath condensate may be useful in monitoring asthma.     

Increased 8-isoprostane and interleukin-6 in breath condensate of obstructive sleep apnea patients

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is characterized by repeated episodes of upper airways obstruction during sleep that result in episodes of hypoxia. An increase of systemic biomarkers of inflammation and oxidative stress has been found in patients with OSA and obesity. DESIGN: The aim of this study was to measure the levels of markers of inflammation (interleukin [IL]-6) and oxidative stress (8-isoprostane) in the exhaled breath condensate of OSA and obese patients. PATIENTS AND METHODS: Eighteen OSA patients (13 men; mean [+/- SEM] age, 44 +/- 7 years), 10 obese subjects (4 men; mean age, 39 +/- 8 years), and 15 healthy age-matched subjects (8 men; mean age, 42 +/- 4 years) were recruited. IL-6 and 8-isoprostane were measured in exhaled breath condensate by a specific enzyme immunoassay kit. Measurements and results: Higher concentrations of IL-6 were found in OSA patients (8.7 +/- 0.3 pg/mL) than in healthy control subjects (1.6 +/- 0.1 pg/mL; p < 0.0001). Obese subjects also had higher levels than healthy control subjects, but lower levels than OSA patients (2.1 +/- 0.2 pg/mL, p < 0.05 and p < 0.0001 respectively). Furthermore, 8-isoprostane levels were found to be higher in OSA patients (7.4 +/- 0.7 pg/mL) than in obese subjects (5 +/- 0.3 pg/mL; p = 0.4) and healthy subjects (4.5 +/- 0.5 pg/mL; p < 0.005). We found a positive correlation between these two markers and neck circumference and apnea/hypopnea index. CONCLUSIONS: These findings suggest that inflammation and oxidative stress are characteristic in the airways of OSA patients but not in obese subjects, and that their levels depend on the severity of the OSA. The measurement of IL-6 and 8-isoprostane levels may prove to be useful in screening and monitoring obese patients who have a high risk of developing OSA.     

Airway inflammation in subjects with gastro-oesophageal reflux and gastro-oesophageal reflux-related asthma

STUDY OBJECTIVES: Asthma and gastro-oesophageal reflux (GER) are both characterized by airway inflammation. DESIGN: The purposes of this work were (i) to study airway inflammation in patients troubled by gastro-oesophageal reflux (GER) and GER associated with asthma, (ii) to ascertain whether GER can aggravate asthma by exacerbating the pre-existing airway inflammation and oxidative stress and (iii) to establish the validity of analysing breath condensate and induced sputum when studying the airways of subjects affected by GER. PATIENT S AND METHODS: We enrolled 14 patients affected by mild asthma associated with GER (40 +/-12 years), nine with mild but persistent asthma (39 +/- 13 years), eight with GER (35 +/- 11 years) and 17 healthy subjects (37 +/- 9 years). Sputum cell counts and concentrations of interleukin-4 (IL-4), IL-6 and 8-isoprostane were measured in breath condensate and supernatant. MEASUREMENTS AND RESULTS: GER-related asthma is characterized by an eosinophilic inflammation, as determined by elevated concentrations of IL-4 in breath condensate and sputum supernatant, and by sputum cell analysis. GER alone presents a neutrophilic pattern of inflammation when determined by elevated concentrations of IL-6 in sputum cell analysis. A concomitant increase has been found in 8-isoprostane in GER associated (or not associated) with asthma. CONCLUSIONS: We conclude that GER is characterized by a neutrophilic airway inflammation and by increased oxidative stress. GER does not however aggravate pre-existing airway inflammation in asthma patients. Determinations of inflammatory and oxidant markers in the breath condensate of subjects with GER reflect these measured in the induced sputum.     

Effect of inhaled corticosteroid treatment on exhaled breath condensate leukotriene E(4) in children with mild asthma

Chronic airway inflammation in children with asthma might be present even in the absence of pathological lung function tests and is known to increase the risk of permanent pulmonary damage. Thus, we aimed at investigating to what extent inflammatory markers such as leukotrienes (LTs) in exhaled breath condensate (EBC) or fractional exhaled nitric oxide (FE(NO)) reflect therapeutic effects in these patients. Fifty steroid-naive patients (aged 8.8 +/- 2.7 years) were included in the study. EBC was collected before and 6 months after therapy with inhaled corticosteroids. LTs were determined by using commercially available ELISA. In addition, FE(NO) was measured by means of a chemiluminescence analyzer. Conventional lung function testing was performed revealing vital capacity, forced expiratory volume, maximum expiratory flow, and specific resistance. In EBC, LTE(4) but not LTB(4) levels significantly decreased after steroid therapy from 45.3 +/- 36.0 pg/mL to 17.2 +/- 11.4 pg/mL (p < 0.0001) concomitant with a slight, but significant improvement of lung function parameters. Mean FE(NO) also indicated therapeutic success; however, in 20 of 50 patients, exhaled NO concentrations were higher after therapy. These findings suggest that LTE(4) in breath condensate may be helpful in latent inflammatory activity in the bronchial mucosa in children with asthma.     

Markers of airway inflammation in primary ciliary dyskinesia studied using exhaled breath condensate

Macroscopically, the airways in primary ciliary dyskinesia (PCD) are inflamed and infected, and the eventual result is bronchiectasis. The measurement of noninvasive markers of inflammation in PCD may allow determination of mechanisms of tissue damage, and even allow monitoring of therapy. The aim of this study was to measure in exhaled breath condensate (EBC) of children with PCD the concentrations of the neutrophil chemoattractants leukotriene (LT) B4 and interleukin (IL)-8 and the marker of oxidative stress 8-isoprostane (8-IP), and to try determining whether these markers can be used to assess mechanisms of airway inflammation in these patients. Concentrations of LTB4, IL-8, and 8-IP in the EBC of 23 PCD and 11 age-matched healthy children were measured using an enzyme immunoassay (EIA). The children also performed spirometry and underwent sputum induction, the latter for differential cell count. The concentrations of 8-IP in EBC of children with stable PCD were significantly increased compared to normal controls (median, 7.8 pg/ml vs. 3.1 pg/ml; P = 0.004). There was no difference in the median concentrations of EBC LTB4 between PCD subjects and healthy controls (28 pg/ml vs. 28 pg/ml; P = 0.5). IL-8 levels were below the detection limit of the assay, and were not analyzed further. There was no correlation between concentrations of either 8-IP or LTB(4) in EBC and forced expired volume in 1 sec in PCD children. Sputum induction was successful in 83% of the subjects; the median induced sputum neutrophil count was 69% (interquartile range, 59.3-73.6). No significant correlation was found between sputum neutrophils and either EBC 8-IP or LTB4 concentrations in PCD children. This study showed that oxidative stress, as reflected by increased exhaled 8-IP concentration, is increased in PCD children. The mechanism of airway neutrophilia is unclear, but is unlikely to be related to increased production of LTB4, at least in stable PCD patients.     

Effect of montelukast on exhaled leukotrienes and quality of life in asthmatic patients

STUDY OBJECTIVES: In some patients with asthma treated with inhaled corticosteroids, suppression of inflammation is incomplete. This may be because the effect of corticosteroids on cysteinyl-leukotriene (cys-LT) biosynthesis is limited. Montelukast is a cys-LT antagonist that significantly improves asthma control in corticosteroid-treated asthmatic patients. However, not all patients treated with cys-LT antagonists show a clinical improvement. DESIGN: We have studied the effect of treatment for 4 weeks with montelukast (10 mg/d) on exhaled cys-LTs and leukotriene B4 (LTB4), exhaled nitric oxide, asthma quality of life (AQL), and respiratory function in patients with stable asthma. SETTING: Asthma clinics in general practice. PATIENTS: We studied 50 patients (30 men; mean +/- SEM age, 53 +/- 2 years) who were treated with inhaled corticosteroids. MEASUREMENTS AND RESULTS: We detected cys-LTs in exhaled breath condensate in 25 of 50 patients; however, in the normal nonasthmatic subjects, cys-LTs were below the limit of detection. After treatment with montelukast, there was a fall in cys-LT concentrations from 14.6 +/- 3.3 to 8.5 +/- 2.6 pg/mL after 2 weeks (p > 0.05) and to 3.9 +/- 1.3 pg/mL after 4 weeks (p < 0.01). Exhaled LTB4 levels were also elevated. After treatment with montelukast, LTB4 levels fell from 33.0 +/- 3.9 to 20.4 +/- 2.5 pg/mL after 2 weeks of treatment (p < 0.05), and to 17.0 +/- 2.2 pg/mL after 4 weeks of treatment (p < 0.01). These changes in exhaled cys-LT and LTB4 were associated with significant improvements in AQL scores. CONCLUSIONS: It appears that in some patients with stable asthma treated with inhaled corticosteroids, the suppression of inflammation is incomplete. Adding a leukotriene receptor antagonist can provide a complementary effect of controlling inflammation, with a significant improvement in quality of life.     

Increased nitrotyrosine in exhaled breath condensate in cystic fibrosis.

Exhaled nitric oxide (ENO), a marker of inflammation in airway diseases is decreased in cystic fibrosis (CF) patients, perhaps because nitric oxide (NO) is metabolized to oxidative end-products. A stable product, 3-nitrotyrosine, may indicate local formation of reactive nitrogen species. Whether NO metabolites in exhaled breath condensate may be increased in CF patients was investigated. The fractional concentration of ENO (Feno), nitrotyrosine and oxides of nitrogen in exhaled breath condensate from 36 stable CF patients were compared to 14 normal subjects using an enzyme immunoassay and fluorescence assay. Nitrotyrosine levels in breath condensate were increased significantly in stable CF patients, compared with normal subjects (25.3 +/- 1.5 versus 6.3 +/- 0.8 ng x mL(-1), p<0.0001). There was an inverse correlation between the levels of nitrotyrosine and the severity of lung disease. Feno levels were significantly lower in CF patients than in normal subjects (4.4 +/- 0.3 versus 5.6 +/- 0.4 (parts per billion), p<0.05). No correlation was found between nitrotyrosine and Feno levels in CF. There was no significant difference in the levels of nitrite and nitrate between CF patients and normals. The elevation in nitrotyrosine may reflect increased formation of reactive nitrogen species such as peroxynitrite or direct nitration by granulocyte peroxidases, indicating increased oxidative stress in airways of cystic fibrosis patients.     

Longitudinal monitoring of lung injury in children after acute chlorine exposure in a swimming pool

RATIONALE: Acute exposure to chlorine gas results in respiratory impairment, but few data are available on the pathobiology of the underlying lung damage. OBJECTIVES: To assess lung function and potential lung damage pathways in the acute phase and longitudinally over a 15-mo follow-up after acute chlorine exposure. METHODS: Ten previously healthy children were accidentally exposed to chlorine gas at a swimming pool because of an erroneous servicing procedure. The fraction of nitric oxide in exhaled air (Fe(NO)), exhaled breath condensate compounds, and serum Clara cell-specific protein CC16 were repeatedly measured. MAIN RESULTS: In the acute phase, all patients had respiratory distress (one child required mechanical ventilation) and reduced lung function (median and interquartile range: FVC, 51 [43-60]% predicted; FEV(1), 51 [46-60]% predicted). This was accompanied by low Fe(NO) (4.7 [3.9-7.9] ppb), high exhaled breath condensate leukotriene B(4) (LTB(4)) levels (24.4 [22.5-24.9] pg/ml), and increased serum CC16 levels (mean +/- SEM, 23.4 +/- 2.5 microg/L). Lung function returned to normal in 15 d (FVC, 97% predicted [82-108], and FEV(1), 92% predicted [77-102]). Fe(NO) reached normal values after 2 mo (12.6 [11.4-15] ppb), whereas LTB(4) levels were still increased (12 [9.3-17.1] pg/ml). CONCLUSION: Children acutely exposed to chlorine in a swimming pool presented a substantial lung function impairment associated with biochemical exhaled breath alterations, represented mainly by an increase in LTB(4) and a reduction in Fe(NO). Although lung function and Fe(NO) improved within a few weeks, the increased levels of exhaled LTB(4) persisted for several months.     

Increased exhaled cysteinyl-leukotrienes and 8-isoprostane in aspirin-induced asthma

The pathogenesis of aspirin-induced asthma (AIA) has not yet been clearly elucidated, although eicosanoid metabolites appear to play an important role. We hypothesized that levels of eicosanoids in exhaled air condensate are abnormal in patients with AIA and that they change in patients receiving steroid therapy. We measured cysteinyl-leukotrienes (cys-LTs), prostaglandin E(2) (PGE(2)), and leukotriene B(4) (LTB(4)), and also 8-isoprostane as a marker of oxidative stress, by enzyme immunoassay in exhaled breath condensate from patients with AIA (17 steroid naive; mean age, 41 +/- 23 years; FEV(1), 63%pred), 26 patients with aspirin-tolerant asthma (ATA) (11 steroid naive; mean age, 47 +/- 18 years; FEV(1), 69%pred), and 16 healthy subjects (mean age, 45 +/- 17 years; FEV(1), 93%pred). Cys-LTs were significantly higher in steroid-naive patients with AIA compared with steroid-naive patients with ATA and healthy subjects (152.3 +/- 30.4 and 36.6 +/- 7.1 versus 19.4 +/- 2.8 pg/ml; p < 0.05 and p < 0.05, respectively). Steroid-naive patients with AIA also had higher levels of 8-isoprostane than normal subjects (131.8 +/- 31.0 versus 21.9 +/- 4.5 pg/ml; p < 0.05). There were significantly lower levels of both cys-LTs and 8-isoprostanes in steroid-treated patients with AIA. There was no difference in either the PGE(2) or LTB(4) level between the patient groups. This is the first study to show that cys-LTs and 8-isoprostanes are elevated in expired breath condensate of steroid-naive patients with AIA, and that cys-LTs are decreased in steroid-treated patients. Exhaled PGE(2) levels are not reduced, so that it is unlikely that a deficiency of PGE(2) is an important mechanism, whereas exhaled LTB(4) levels are unchanged, indicating an abnormality beyond 5-lipoxygenase.     

Production of the potent neutrophil chemokine, growth-related protein alpha (GROalpha), is not elevated in cystic fibrosis children

Progressive neutrophil-mediated lung damage causes much of the morbidity and mortality in cystic fibrosis (CF). Neutrophil chemoattractants implicated in CF include interleukin (IL-)8, tumour necrosis factor (TNFalpha) and leukotriene (LT)B4, but growth-related protein alpha (GROalpha), a highly potent neutrophil chemokine, has not been investigated. Atopic status has been considered to contribute to the marked heterogeneity of pulmonary disease in CF. We hypothesized that GROalpha may be produced in biologically-significant amounts in the CF lung, and that enhanced production of GROalpha, IL-8 or LTB4 may contribute to the poorer lung function seen in atopic CF patients compared to non-atopic CF patients. GROalpha, IL-8 and LTB4 levels in the sputum of atopic and non-atopic CF patients were assessed by immunoassays, and GROalpha and IL-8 levels were also assessed in the plasma of CF patients and normal controls. As expected, there were high levels of IL-8 and LTB4 in most CF sputum samples, and IL-8 levels were higher in CF plasma than in control plasma (P=0.02). In contrast, GROalpha was undetectable (< 5 pg ml(-1)) in the sputum of 21 out of 25 CF patients, with low levels (range 144-825 pg ml(-1)) in the remainder, and median levels of GROalpha in CF plasma (33 pg ml(-1), n=24) were not significantly different from controls (34 pg ml(-1), n=25). Lung function [forced expiratory volume in 1 sec (FEV1) and forced vital capacity (FVC)] was significantly poorer in atopic CF compared to non-atopic CF patients (P<0.02), but sputum levels of GROalpha, IL-8 and LTB4 were not different between the subgroups. Our results suggest that unlike LTB4 and IL-8, GROalpha does not contribute to neutrophilic inflammation in the CF lung, and other factors must determine the impaired lung function observed in atopic CF patients. These results may have important implications in the development of chemokine receptor antagonists as novel anti-inflammatory agents in CF.     

Inflammatory mediators in exhaled breath condensate of children with obstructive sleep apnea syndrome

BACKGROUND: Upper airway inflammation is now recognized in adults with obstructive sleep apnea (OSA) syndrome. However, the role played by eicosanoids such as leukotrienes and prostaglandins is unclear. OBJECTIVE: To investigate whether eicosanoids are measurable in exhaled breath condensate (EBC), and to determine whether differences in these inflammatory mediators emerge among children with and without sleep-disordered breathing (SDB). METHODS: EBC was collected from 50 consecutive snoring children undergoing overnight polysomnography for suspected SDB, and from 12 nonsnoring control subjects. Prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and cysteinyl leukotrienes (cys-LTs: leukotriene C4 [LTC4]/leukotriene D4 [LTD4]/leukotriene E4 [LTE4]) EBC levels were analyzed using enzyme-linked immunosorbent assay. RESULTS: LTB4 levels were elevated in children with an apnea-hypopnea index (AHI) > 5/h (SDB; 97.6 +/- 6.3 pg/mL) compared to children with an AHI < 5/h (mild SDB; 66.4 +/- 19.1 pg/mL; p < 0.01) and control subjects (27.8 +/- 3.7 pg/mL; p < 0.01). Similarly, cys-LT (LTC4/LTD4/LTE4) concentrations were also increased in SDB (45.1 +/- 10.6 pg/mL in SDB vs 27.6 +/- 8.3 pg/mL in mild SDB, and 15.7 +/- 7.6 pg/mL in control subjects; p < 0.01). In contrast, PGE2 concentrations were similar among the three groups. CONCLUSIONS: Inflammatory mediators such as leukotrienes and prostaglandins can be readily quantified in EBC collected from the upper airway of children. Disease severity-dependent increases in leukotriene concentrations (LTB4 and LTC4/LTD4/LTE4) emerge among children and may serve as a noninvasive tool in the clinical assessment of these children.     

Biomarkers of neutrophilic inflammation in exhaled air of cystic fibrosis children with bacterial airway infections

Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) are inflammatory mediators involved in the neutrophil response to pulmonary bacterial colonization in cystic fibrosis (CF). The aim of this study was to investigate whether the LTB(4) and IL-8 levels in exhaled breath condensate (EBC) could be related to the type of bacterial colonization in CF patients. The pH level in EBC was analyzed as an estimate of airway acidification. Forty children were evaluated: 10 CF patients with P. aeruginosa, 10 CF patients with S. aureus, 10 not colonized CF patients, and 10 healthy children. LTB(4) and IL-8 in EBC were analyzed by specific enzyme immunoassay kits (EIA). The pH of EBC was measured with a pH-meter after deareation by bubbling with argon. Exhaled LTB(4) was higher in CF children with P. aeruginosa compared to those with S. aureus (P < 0.01), not colonized (P < 0.001), and healthy children (P < 0.01). Exhaled IL-8 was elevated in CF patients colonized by P. aeruginosa compared with other subgroups (vs. not colonized, P < 0.05; vs. healthy children, P < 0.001). IL-8 levels were higher in CF children with S. aureus than in healthy children (P < 0.05). There was an increase in IL-8 levels in not colonized CF patients compared with healthy children (P < 0.05). EBC pH was higher in healthy children compared to CF patients not colonized (P < 0.05). Our data suggest that EBC is suitable for evaluating neutrophil inflammatory mediators (LTB(4), IL-8, and pH) involved in the response to pulmonary bacterial colonization in CF children.     

Increased nitrosothiols in exhaled breath condensate in inflammatory airway diseases

Nitrosothiols (RS-NOs) are formed by interaction of nitric oxide (NO) with glutathione and may limit the detrimental effect of NO. Because NO generation is increased in airway inflammation, we have measured RS-NOs in exhaled breath condensate in patients with asthma, cystic fibrosis, or chronic obstructive pulmonary disease (COPD). We also measured exhaled NO and nitrite (NO(2-)) in the same subjects. RS-NOs were detectable in exhaled breath condensate of all subjects. RS-NOs were higher in subjects with severe asthma (0.81 +/- 0.06 microM) when compared with normal control subjects (0.11 +/- 0.02 microM, p < 0.01) and with subjects with mild asthma (0.08 +/- 0.01 microM, p < 0.01). Elevated RS-NOs values were also found in patients with cystic fibrosis (0.35 +/- 0.07 microM, p < 0.01), in those with COPD (0.24 +/- 0.04 microM, p < 0.01) and in smokers (0.46 +/- 0.09 microM, p < 0.01). In current smokers there was a correlation (r = 0.8, p < 0.05) between RS-NOs values and smoking history (pack/year). We also found elevated concentrations of NO(2-) in patients with severe asthma, cystic fibrosis, or COPD, but not in smokers or patients with mild asthma. This suggests that exhaled NO(2-) is less sensitive than exhaled RS-NOs. This study has shown that RS-NOs are detectable in exhaled breath condensate of healthy subjects and are increased in patients with inflammatory airway diseases. As RS-NOs concentrations in exhaled breath condensate vary in the different airway diseases and increase with the severity of asthma, their measurement may have clinical relevance as a noninvasive biomarker of nitrosative stress.     

Increased 8-isoprostane, a marker of oxidative stress, in exhaled condensate of asthma patients.

Oxidative stress has an important role in the pathogenesis of asthma. 8-Isoprostane is a prostaglandin (PG)-F2-like compound belonging to the F2 isoprostane class that is produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid. 8-Isoprostane is a biomarker of oxidative stress, and its concentration is increased in the bronchoalveolar lavage fluid of patients with interstitial lung diseases. We measured 8-isoprostane concentrations in exhaled breath condensate in healthy subjects and in patients with mild (steroid naive, n = 12), moderate (inhaled steroid treatment, n = 17), and severe asthma (oral steroid treatment, n = 15). We also measured exhaled carbon monoxide (CO) and nitric oxide (NO), which may also reflect oxidative stress in the airways. 8-Isoprostane was detectable in breath condensate of normal subjects (15.8 +/- 1.6 pg/ml), and was increased in the breath condensate of patients with mild (33.7 +/- 2.8, p < 0.001), moderate (38.3 +/- 3.7 pg/ml, p < 0. 001), and severe asthma (48.9 +/- 5.0 pg/ml, p < 0.001). There was a positive correlation (r = 0.68, p < 0.05) of 8-isoprostane with NO, but not with CO, in the exhaled air of patients with mild asthma, but not in that of patients with moderate or severe asthma. There was no correlation between 8-isoprostane and lung function tests in any group of patients. Our study shows that oxidative stress is increased in asthmatic subjects as reflected by 8-isoprostane concentrations in breath condensate.     

Exhaled breath condensate cytokines and pH in pediatric asthma and atopic dermatitis

Some studies have proposed exhaled breath condensate (EBC) as a noninvasive tool for monitoring airway inflammation in children. Moreover, atopic dermatitis (AD) has been considered a risk factor for the development of asthma. This study was designed to assess the EBC pH and the exhaled concentration of cytokines produced by T-helper (Th) 1, Th2, and T regulatory cells in asthmatic children and AD and to verify if their concentrations are affected by a short course of treatment with inhaled corticosteroids (ICS). We assessed the mean levels of pH, interferon (IFN) gamma, interleukin (IL)-4, and IL-10 in EBC of children with asthma (n=20) and AD (n=12) and healthy controls (n=20) by enzyme-linked immunosorbent assay (ELISA). Variations of pH and cytokine concentration in response to ICS (flunisolide, 500 microg/day, for 2 weeks), were also investigated in asthmatic patients. We found that the mean condensate pH value in patients with asthma and AD was significantly lower when compared with that of controls (6.9+/-0.2 and 7.0+/-0.2 versus 7.4+/-0.4; p<0.0001) and it significantly increased in asthmatic patients after treatment (7.2+/-0.2 versus 6.9+/-0.2; p=0.003). In addition, the IL-4/IFN-gamma ratio was significantly higher in children with asthma and in those with AD when compared with controls (9.72+/-2.00 and 9.70+/-2.0 versus 8.04+/-2.6; p<0.001) and that it decreased in asthmatic patients after ICS (6.4+/-5.4 versus 9.72+/-2.00; p<0.01). We observed that exhaled IL-10 levels were significantly higher in children with asthma compared with those of controls (18.8+/-8.9 versus 4.2+/-1.0; p<0.002). IL-10 did not significantly increase after treatment with steroids. No such finding was documented in children with AD. Our data suggest that EBC IL-10 levels are different in asthmatic patients compared with healthy children, but they are insensitive markers in monitoring therapy with ICS. Moreover, children with AD show an EBC pH and an exhaled pattern of Th2/Th1 cytokines similar to that of asthmatic patients.