Vanessa Brown

Exhaled nitric oxide correlates with airway eosinophils in childhood asthma

Background: Exhaled nitric oxide has been proposed as a marker for airway inflammation in asthma. The aim of this study was to compare exhaled nitric oxide levels with inflammatory cells and mediators in bronchoalveolar lavage fluid from asthmatic and normal children. Methods: Children were recruited from elective surgical lists and a non-bronchoscopic bronchoalveolar lavage (BAL) was performed after induction of anaesthesia. Exhaled nitric oxide (parts per billion) was measured by two techniques: tidal breathing and restricted breath. Results: Median (interquartile range) exhaled nitric oxide measured by restricted breath was increased in asthmatics compared with normal children (24.3 (10.5–66.5) v 9.7 (6.5–16.5), difference between medians 14.6 (95% CI 5.1 to 29.9), p=0.001). In asthmatic children exhaled nitric oxide correlated significantly with percentage eosinophils (r=0.78, p<0.001 (tidal breathing) and r=0.78, p<0.001 (restricted breath)) and with eosinophilic cationic protein (r=0.53, p<0.01 (restricted breath)), but not with other inflammatory cells in the BAL fluid. The area under the receiver operator characteristic curves for the prediction of the presence of eosinophilic airways inflammation by exhaled nitric oxide (tidal and restricted) was 0.80 and 0.87, respectively. Conclusions: Exhaled nitric oxide correlates closely with percentage eosinophils in BAL fluid in asthmatic children and is therefore likely to be a useful non-invasive marker of airway inflammation.

Simvastatin decreases lipopolysaccharide-induced pulmonary inflammation in healthy volunteers.

RATIONALE Simvastatin inhibits inflammatory responses in vitro and in murine models of lung inflammation in vivo. As simvastatin modulates a number of the underlying processes described in acute lung injury (ALI), it may be a potential therapeutic option. OBJECTIVES To investigate in vivo if simvastatin modulates mechanisms important in the development of ALI in a model of acute lung inflammation induced by inhalation of lipopolysaccharide (LPS) in healthy human volunteers. METHODS Thirty healthy subjects were enrolled in a double-blind, placebo-controlled study. Subjects were randomized to receive 40 mg or 80 mg of simvastatin or placebo (n = 10/group) for 4 days before inhalation of 50 microg LPS. Measurements were performed in bronchoalveolar lavage fluid (BALF) obtained at 6 hours and plasma obtained at 24 hours after LPS challenge. Nuclear translocation of nuclear factor-kappaB (NF-kappaB) was measured in monocyte-derived macrophages. MEASUREMENTS AND MAIN RESULTS Pretreatment with simvastatin reduced LPS-induced BALF neutrophilia, myeloperoxidase, tumor necrosis factor-alpha, matrix metalloproteinases 7, 8, and 9, and C-reactive protein (CRP) as well as plasma CRP (all P < 0.05 vs. placebo). There was no significant difference between simvastatin 40 mg and 80 mg. BALF from subjects post-LPS inhalation induced a threefold up-regulation in nuclear NF-kappaB in monocyte-derived macrophages (P < 0.001); pretreatment with simvastatin reduced this by 35% (P < 0.001). CONCLUSIONS Simvastatin has antiinflammatory effects in the pulmonary and systemic compartment in humans exposed to inhaled LPS.

Neutrophil apoptosis, proinflammatory mediators and cell counts in bronchiectasis

Background: Lower airway secretions from patients with bronchiectasis show inflammatory cell infiltration and increased proinflammatory mediators. The aim of this study was to investigate the effects of antibiotic treatment for exacerbations on neutrophil apoptosis and necrosis. Methods: Sputum was induced from 15 subjects with idiopathic bronchiectasis at the beginning of an acute exacerbation and after intravenous antibiotic treatment. Neutrophil apoptosis and necrosis were assessed using flow cytometry and morphology and the supernatant was analysed for concentrations of inflammatory mediators. Results: Neutrophil numbers (×106 cells/g sputum) in sputum were significantly greater on day 0 than on day 14 (median difference (95% confidence interval (CI)) 5.14 (1.27 to 8.46), p = 0.02). Controls had a significantly higher percentage of sputum macrophages than patients with bronchiectasis (day 0, 1.35 (95% CI 0.48 to 2.89), p = 0.004; day 14, 1.09 (95% CI 0.26 to 2.86), p = 0.02). The concentrations of tumour necrosis factor α (pg/ml), interleukin 8 (ng/ml), and neutrophil elastase (ng/ml) in sputum supernatant were significantly reduced on day 14 compared with day 0 (median difference −94 (95% CI −158 to −27), p = 0.005; −106 (95% CI −189 to −50), p = 0.0006; and −73 451 (95% CI −135 495 to −12 303), p = 0.02 respectively). Patients with bronchiectasis had a significantly lower percentage of cells which were neither apoptotic nor necrotic than healthy controls (both days, −38.8 (95% CI −49.6 to −8.5), p = 0.002; −45.0 (95% CI −58.0 to −34.1), p = 0.0003, respectively), and on day 14 they had a significantly higher percentage of secondary necrotic cells than healthy controls (40 (95% CI 11.6 to 57.5), p = 0.004). Conclusions: This study shows that antibiotic treatment affects concentrations of proinflammatory mediators and cell death and clearance may be altered in bronchiectasis.

Flow-cytometric analysis of basophil activation: inhibition by histamine at conventional and homeopathic concentrations.

Human basophils play a pivotal role in the pathogenesis of chronic allergic diseases such as asthma. Activation of basophils by cross-linking of IgE induces fusion of the cytoplasmic granules with the plasma membrane and the subsequent release of potent mediators including histamine. Basophil activation can be quantified by the measurement of secreted histamine [1] and by direct microscopic examination of the percentage of degranulated cells [2]. More recently, flow cytometry has been used to measure basophil activation using the cell surface marker CD63, in conjunction with antiIgE [3]. CD63 is expressed on intracytoplasmic granules of resting basophils and only weakly expressed on the outside membrane (<5%) [4]. However, basophil activation leads to the upregulation of CD63 on the cell surface, which can be easily quantified using flow cytometry. Both pharmacological concentrations and high dilutions of histamine have been reported to modulate anti-IgE induced basophil degranulation [5]. The aim of this study was to confirm that a 2-colour (anti-IgE FITC and anti-CD63 R-PE) flow cytometric method can quantify basophil activation and to investigate the effects of histamine on basophil activation.

T cell cytokine profiles in childhood asthma

Background: An imbalance of T cell subsets in asthma with a predominance of Th2 type cells has been proposed. The aim of this study was simultaneously to detect surface markers and intracellular production of cytokines in T cells from the airways of children with and without asthma. Methods: Bronchoalveolar lavage (BAL) fluid was obtained by wedging a suction catheter into the distal airway immediately before elective surgery. Cells were stimulated with phorbol 12-myristrate 13-acetate (PMA) and ionomycin and intracytoplasmic cytokine retention was achieved using monensin. The cells were stained with the relevant antibodies and analysed by flow cytometry. Results: No statistical difference was observed between children with atopic asthma, atopic non-asthmatic subjects, and normal controls in the percentage of CD3+ cells producing interleukin (IL)-2 or IL-4. Interferon (IFN)γ+ T cells were, however, present in a much higher percentage than either IL-2 or IL-4 positive cells. The percentage of IFNγ+ T cells was significantly increased in subjects with atopic asthma (median 71.3%, interquartile range (IQR) 65.1–82.2, n=13) compared with both atopic non-asthmatic subjects (51.9%, IQR 37.2–70.3, n=12), p<0.05 and normal controls (58.1%, IQR 36.1–66.1, n=23), p<0.01. Conclusions: These findings indicate that IFNγ producing T cells are more abundant in the airways of children with atopic asthma than in atopic non-asthmatic subjects and controls. The proinflammatory activities of IFNγ may play an important role in the pathogenesis of childhood asthma and may suggest that asthma is not simply a Th2 driven response.

Outgrown asthma does not mean no airways inflammation

Although some asthmatic children seem to recover from their asthma, 30–80% develop asthma again in later life. The underlying risk factors are unknown. The hypothesis for this study was that children with apparently outgrown asthma would have underlying airway inflammation. Nonbronchoscopic bronchoalveolar lavage was performed on normal children (n=35) and children who had wheezed previously (n=35). Eosinophils were raised in the lavage fluid of atopic children who had apparently outgrown asthma (median (interquartile range) 0.36 (0.05–0.74) compared to controls 0.10 (0–0.18), p=0.002). There was no relationship between length of remission and degree of airways eosinophilia. Thus, there is persistent airways inflammation in some children with outgrown asthma and this may be a risk factor for future relapse.

Dysregulated apoptosis and NFκB expression in COPD subjects

BackgroundThe abnormal regulation of neutrophil apoptosis may contribute to the ineffective resolution of inflammation in chronic lung diseases. Multiple signalling pathways are implicated in regulating granulocyte apoptosis, in particular, NFκB (nuclear factor-kappa B) signalling which delays constitutive neutrophil apoptosis. Although some studies have suggested a dysregulation in the apoptosis of airway cells in chronic obstructive pulmonary disease (COPD), no studies to date have directly investigated if NFκB is associated with apoptosis of airway neutrophils from COPD patients. The objectives of this study were to examine spontaneous neutrophil apoptosis in stable COPD subjects (n = 13), healthy smoking controls (n = 9) and non-smoking controls (n = 9) and to investigate whether the neutrophil apoptotic process in inflammatory conditions is associated with NFκB activation.MethodsAnalysis of apoptosis in induced sputum was carried out by 3 methods; light microscopy, Annexin V/Propidium iodide and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method. Activation of NFκB was assessed using a flow cytometric method and the phosphorylation state of IκBα was carried out using the Bio-Rad Bio-Plex phosphoprotein IκBα assay.ResultsFlow cytometric analysis showed a significant reduction in the percentage of sputum neutrophils undergoing spontaneous apoptosis in healthy smokers and subjects with COPD compared to non-smokers (p < 0.001). Similar findings were demonstrated using the Tunel assay and in the morphological identification of apoptotic neutrophils. A significant increase was observed in the expression of both the p50 (p = 0.006) and p65 (p = 0.006) subunits of NFκB in neutrophils from COPD subjects compared to non-smokers.ConclusionThese results demonstrate that apoptosis is reduced in the sputum of COPD subjects and in healthy control smokers and may be regulated by an associated activation of NFκB.

Inflammatory mediators in bronchoalveolar lavage samples from children with and without asthma

We investigated whether eosinophils and mast cells, found in the airways of children with wheeze, were activated during relatively asymptomatic periods.

Antioxidants and oxidative stress in BAL fluid of atopic asthmatic children

Earlier studies in adults have indicated that increased oxidative stress may occur in the blood and airways of asthmatic subjects. Therefore the aim of this study was to compare the concentrations of antioxidants and protein carbonyls in bronchoalveolar lavage fluid of clinically stable atopic asthmatic children (AA, n = 78) with our recently published reference intervals for nonasthmatic children (C, n = 124). Additionally, lipid peroxidation products (malondialdehyde) in bronchoalveolar lavage fluid and several antioxidants in plasma were determined. Bronchoalveolar lavage concentrations (median and interquartile range) of ascorbate [AA: 0.433 (0.294–0.678) versus C: 0.418 (0.253-0.646) μmol/L], urate [AA: 0.585 (0.412–0.996) versus C: 0.511 (0.372–0.687) μmol/L], α-tocopherol [AA: 0.025 (0.014–0.031) versus C: 0.017 (0.017–0.260) μmol/L], and oxidized proteins as reflected by protein carbonyls [AA: 1.222 (0.970–1.635) versus C: 1.243 (0.813–1.685) nmol/mg protein] were similar in both groups (p > 0.05 in all cases). The concentration of protein carbonyls correlated significantly with the number of eosinophils, mast cells, and macrophages in AA children only. Concentrations of oxidized proteins and lipid peroxidation products (malondialdehyde) correlated significantly in AA children (r = 0.614, n = 11, p = 0.044). Serum concentrations of ascorbate, urate, retinol, α-tocopherol, β-carotene, and lycopene were similar in both groups whereas α-carotene was significantly reduced in asthmatics. Overall, increased bronchoalveolar lavage eosinophils indicate ongoing airway inflammation, which may increase oxidatively modified proteins as reflected by increased protein carbonyl concentrations.

Cytokine concentrations and neutrophil elastase activity in bronchoalveolar lavage and induced sputum from patients with cystic fibrosis, mild asthma and healthy volunteers

BACKGROUND Induced sputum (IS) has been proposed as a non-invasive alternative to bronchoalveolar lavage (BAL) for the assessment and monitoring of airways inflammation. The aim of this study was to compare both methods in patients with cystic fibrosis (CF). The possible differences between subjects with CF, mild asthma and healthy volunteers (HV) was also assessed. METHOD In a single centre, randomised, two way crossover study, 11 patients with CF, 9 mild asthmatics (MA) and 11 HV underwent BAL and hypertonic saline induction on consecutive days. Free neutrophil elastase (NE), neutrophil elastase/alpha(1)-anti-trypsin complex (NE-AAT), tumour necrosis factor receptor (p55) and interleukin-8 (IL-8) were measured in cell free supernatants. RESULTS Three CF patients reported serious adverse events following BAL. NE was usually undetectable in both IS or BAL samples and NE-AAT concentrations did not differ consistently between the two sampling methods. IL-8 and p55 levels in the CF patients tended to be higher in IS samples compared with BAL samples (median 19,860 vs. 3,855 pg/ml and 2.55 vs. 0.29 ng/ml, respectively). There was a significant difference in mean p55 concentrations between CF, MA and HV in IS samples (P=0.003) but not in BAL samples (P=0.36). The difference in mean IL-8 concentrations in IS samples between subject groups was statistically different (P=0.023). CONCLUSIONS IS samples can be safely obtained from CF patients. Analysis of IS samples can help to characterize the inflammatory process in the airways of CF patients. The serious adverse events following BAL in 3 CF patients highlight an inherent risk associated with this procedure.