Use of induced sputum to examine airway inflammation in childhood asthma

Airway inflammation is important in the pathogenesis of asthma, during which it may lead to symptomatic exacerbations and increases in asthma severity, as well as contribute to future decline in asthma status. The use of induced sputum has emerged as an important and useful technique to study airway inflammation. It has particular advantages in the study of childhood asthma because it is noninvasive and allows samples to be collected on repeated occasions in children over 7 years of age. The results of cell counts are reliable when the sputum is processed in a standardized manner involving selection from saliva, cell dispersion, and quantitative cytology. Children with asthma have increased eosinophils and mast cells, which may persist even with high doses of inhaled corticosteroid therapy. During a severe exacerbation of asthma, there is an intense and heterogeneous inflammatory response involving eosinophil and neutrophil accumulation and activation. Characterization of the relevance of airway inflammation in children with asthma is important.     

Comparison between eosinophilic markers in induced sputum and blood in asthmatic patients

BACKGROUND: The usefulness and safety of the analysis of blood inflammatory markers in asthma are widely recognized. Recently, the analysis of induced sputum has been proposed as a safe, non-invasive tool in the study of airway inflammation in asthma. OBJECTIVE: Our aim was to test whether sputum analysis is more useful than blood analysis in the evaluation of airway inflammation in untreated and treated asthmatic patients. METHODS: Twelve untreated patients with mild to moderate asthma underwent a methacholine challenge test, sputum induction and blood sampling. A group of 14 normal subjects was also evaluated for baseline comparison. The same evaluation was repeated after 3 months of budesonide treatment. Before and after treatment, we tested the relationship of eosinophilic markers in induced sputum and blood with clinical and functional data. We also compared eosinophilic markers in induced sputum with the same markers in blood. RESULTS: Untreated patients showed a significant relationship between sputum eosinophils and symptom score, and between sputum eosinophilic cationic protein and symptom score, FEV1 and PD20FEV1. No relationship between blood eosinophilic markers and clinical or functional data was observed. In budesonide-treated patients, both sputum and blood eosinophils were significantly lower than in untreated patients, but eosinophil decrease was greater in sputum than in blood. Sputum eosinophilic proteins were also significantly lower in treated patients, whereas serum eosinophilic proteins were low at baseline and remained unchanged after treatment. Sputum eosinophilic markers were lower in normal subjects than in both untreated and treated patients, while blood eosinophils, but not serum eosinophilic cationic protein, were lower in normals than in untreated patients. CONCLUSIONS: The analysis of induced sputum is more useful than the analysis of blood in the evaluation of asthma severity and of the effect of glucocorticoid treatment in patients with mild to moderate asthma.     

An inhaled steroid improves markers of airway inflammation in patients with mild asthma

Airway inflammation can be demonstrated in mildly asthmatic patients who are not treated with inhaled steroids. Current guidelines recommend that inhaled steroids should be introduced in mild asthmatics who use an inhaled beta2-agonist more than once daily. It was postulated that inhaled steroids can have anti-inflammatory effects in patients with even milder disease. The effect of 4 weeks of treatment with budesonide (800 microg twice daily by Turbohaler) was studied in 10 steroid-naive mildly asthmatic patients (forced expiratory volume in one second (FEV1) = 96+/-1.4% predicted) who required an inhaled beta2-agonist less than one puff daily, in a double-blind, placebo-controlled, crossover fashion. Spirometry, exhaled nitric oxide (NO), bronchial responsiveness (provocative concentration causing a 20% fall in FEV1 (PC20)), and sputum induction were performed before and after each treatment period. Following budesonide treatment, there were significant improvements in FEV1, and PC20, in association with a significant reduction in the percentage of eosinophils in induced sputum. Exhaled NO levels tended towards reduction, but the change was nonsignificant. There were also nonsignificant reductions in sputum eosinophil cationic protein and tumour necrosis factor-alpha levels. In conclusion inhaled budesonide can lead to improvements in noninvasive markers of airway inflammation, in association with a small improvement in lung function, even in mildly asthmatic patients who require an inhaled beta2-agonist less than once daily. This suggests a potential benefit of inhaled corticosteroids, even in relatively asymptomatic asthma.     

Clinical value of evaluating "markers" of cellular inflammation in monitoring bronchial asthma

In the base of bronchial asthma is a chronic inflammatory process which takes place in bronchial mucous membrane. The main cells of allergic reaction are: mastocytes., basophils, eosinophiles and others. There are many factors released from them in various periods of allergic inflammation. Evaluation of a concentration of these factors may be very usefull in diagnostic and treatment of bronchial asthma.     

Some serum activity markers of airways inflammation in difficult-to-control asthma patients

The main aim of the present study was a search for a characteristic serum marker of inflammatory activity in the airways of asthmatics with difficult-to-control disease. Therefore, serum levels of interleukin-4 (IL-4), serum low-affinity Fc Epsilon Receptor II (sFcER II), Interferon-gamma (INF-gamma) Immunoglobulin-E (IgE), Interleukin-2 (IL-2), serum Interleukin Receptor 2 (sIL-2R) and Intercellular Adhesion Molecule-1 (sICAM-1) were measured in 2 groups of asthmatics: 1-26 patients with difficult-to-control asthma (DTCA), 2-22 asthmatics, minimally symptomatic (MSA). RESULTS: No significant difference in either measured parameters between the DTCA and MSA group in peripheral blood samples was found. CONCLUSION: The above mentioned serum markers of T- and B-cell activation as well as the serum ICAM-1 level are not sensitive enough to determine the type, activity and severity of the inflammatory process in the asthmatic airways.     

Airway mucosal inflammation even in patients with newly diagnosed asthma

We have studied bronchial biopsies from 14 patients with newly diagnosed asthma (four men and 10 women), who had had asthma symptoms, on average, 7.4 months (range, 2 to 12 months) and from four control subjects. The patients had not received corticosteroids, disodium cromoglycate, or theophylline before the study. The bronchial biopsies were taken, using a rigid-tube bronchoscope under local anaesthesia, from two different airway levels: (1) inside the right upper lobe bronchus, and (2) at the opening of the right middle lobe. The specimens were prepared for both light and electron microscopy. The use of Slot grids 1 x 2 mm enabled a large area of the thin sections to be photographed and analyzed by applying a graphic Autocad program. There was an increase in the numbers of mast cells (p < 0.001), eosinophils (p < 0.05), lymphocytes (p < 0.05), and macrophages (p < 0.05) in the epithelium of patients with newly diagnosed asthma as compared with those in control subjects. In the lamina propria, these asthmatic patients had more eosinophils (p < 0.001), lymphocytes (p < 0.001), macrophages (p < 0.001), and plasma cells (p < 0.001) than did the control subjects. We conclude that, in asthma, an airway inflammatory process is present even at a clinically early stage of the disease. In the asthmatic airways, there are signs of a general inflammatory response caused by more than one cell type.     

Impaired perception of dyspnea in patients with severe asthma. Relation to sputum eosinophils

Poor dyspnea perception might be a risk factor for developing asthma exacerbations. We investigated whether severe asthmatics with recurrent exacerbations (brittle asthma) have different dyspnea perception and sputum cells compared with equally severe, but stable asthmatics, or patients with mild steroid-naive asthma. Fifteen brittle asthmatics (13 female, median age 28 yr [range, 20 to 47 yr]), 15 matched severe-stable asthmatics (14 female, median age 26 yr [range, 17 to 52 yr]), and 11 mild asthmatics (8 female, median age 25 yr [range, 19 to 43 yr]) underwent inhalation tests with methacholine (MCh), and hypertonic saline combined with sputum induction. Dyspnea was assessed by Borg and Visual Analogue Scale (VAS), plotted against the percent fall in FEV1, and expressed as the slope of the regression line (Slope-Borg and Slope-VAS). The brittle and stable asthmatics had poorer perception than patients with mild asthma (Slope-Borg [p = 0.036], Slope-VAS [p < 0.001] for MCh). In patients with brittle asthma the perception was less as compared with severe-stable asthma (Slope-Borg for MCh: p = 0.05). In the severe asthmatics there was an inverse correlation between sputum eosinophilia and Slope-Borg and Slope-VAS (R = -0.55, p = 0. 002 and R = -0.37, p = 0.049), whereas this correlation was a positive one in the mild asthmatics (R = 0.79, p = 0.012 and R = 0. 67, p = 0.05). In conclusion, patients with severe asthma, particularly those with recurrent exacerbations, have blunted perception of dyspnea, which is related to the degree of sputum eosinophilia. This suggests that increased sputum eosinophilia is an indicator of clinical instabililty, and that eosinophilic airways inflammation might affect dyspnea perception in severe asthma.     

The effect of theophylline on blood and sputum eosinophils and ECP in patients with bronchial asthma

It was recently reported that theophylline has an anti-inflammatory and bronchodilating effect on bronchial asthma. Accordingly, to examine the anti-inflammatory effect of theophylline on asthma, especially its effect on eosinophil activation, a sustained-release theophylline preparation (Theolong) was administered (daily dose: 400 mg) to 18 patients with mild to moderate bronchial asthma. This was done in order to study the preparation's effects on lung function, blood and sputum eosinophils and ECP four weeks pre- and post-administration. Lung function was determined by spirometry and sputum by induced sputum. Blood and sputum ECP levels were determined using an ECP RIA kit. In lung function, there were no differences in vital capacity (VC) or in forced expiratory volume 1 s (FEV 1.0) pre- and post-administration. There were also no differences in the number of blood and sputum eosinophils, but serum and sputum ECP levels decreased. Theophylline is thus expected to exert an inhibitory effect on eosinophil activation and it is suggested as an effective therapeutic drug for bronchial asthma.     

Induced sputum, bronchoalveolar lavage and blood from mild asthmatics: inflammatory cells, lymphocyte subsets and soluble markers compared

Airway inflammation in asthma can be measured directly by invasive bronchoalveolar lavage (BAL), directly and relatively noninvasively by induced sputum and indirectly from peripheral blood. We compared cellular and fluid phase indices of inflammation in induced sputum, BAL and blood from 11 adults with mild stable asthma. On one day, induced sputum selected from saliva was collected and on the next, blood and BAL. Median results of sputum compared with BAL showed a higher number of nonsquamous cells (53 versus 0.8 x 10(6) cells x mL(-1), p=0.003), more neutrophils (34.3 versus 1.0%, p<0.001), CD4+ and CD19+ T-cells (76.5 versus 54.7%, p=0.01 and 5.2 versus 1.1%, p=0.03, respectively), fewer macrophages (603 versus 95.0%, p=0.002) and markedly higher levels of eosinophil cationic protein (ECP) (264 versus 2.0 microg x L(-1), p<0.001), tryptase (17.6 versus 2.2 UI x L(-1), p<0.001) and fibrinogen (1,400 versus 150 microg x L(-1), p=0.001). Sputum and BAL neutrophils and CD4+ T-cells were strongly correlated. Sputum and BAL differed from blood by having higher proportions of T-cells (94.9 and 98.9% versus 87.7%, p=0.002) and lower proportions of CD19+ T-lymphocytes (p=0.04 and 0.006). Sputum also differed from blood by having higher proportions of CD4+ T-cells (76.5 versus 51.4%, p=0.001), lower proportions of CD8+ cells (24.0 versus 403%, p=0.04) and a higher CD4+/CD8+ ratio (3.3 versus 1.4, p=0.01). We conclude that in mild asthmatics, sputum, bronchoalveolar lavage and blood measure different compartments of inflammation. Induced selected sputum has the advantage over bronchoalveolar lavage of higher density of cell recovery and stronger signal for fluid-phase markers.     

Neutrophil collagenase in sputum from patients with cystic fibrosis

The potential role of neutrophil elastase in causing lung damage and exacerbating the inflammatory response in cystic fibrosis (CF) has received considerable attention. Although another potent neutrophil-derived enzyme, collagenase, is implicated in tissue destruction in several interstitial lung disorders, there has been no reference to this enzyme in CF. The objective of this study was to determine whether neutrophil collagenase is present in active form in CF sputum and, if so, whether it is related to disease severity. High levels of active collagenase were detected in sputum from patients with CF, and the majority of the enzyme present was of neutrophil origin. In a group of 16 patients with CF, negative relations between sputum collagenase activity and Shwachman score (r = -0.55, p < 0.05) and FEV1 (r = -0.59, p < 0.02) were noted, indicating an association between high collagenase activity and severity of disease. A positive correlation was observed between sputum collagenase and elastase activity (r = 0.62, p < 0.05). These results suggest that both neutrophil elastase and collagenase may play a significant role in lung destruction in CF.     

Isolation of pathogenic bacteria from induced sputum from hospitalized children with pneumonia in Bangladesh

A high-performance liquid chromatographic system, combining solid-phase extraction and automated precolumn derivatization is described for the routine determination of methotrexate in human plasma. The sample extraction and elution onto the analytical column were performed automatically and concomitantly using the column-switching technique and a protein-coated precolumn. Cerium (IV) trihydroxyhydroperoxide (CTH) was introduced as a packed oxidant before the analytical column for the conversion of methotrexate into highly fluorescent products. The oxidative-cleavage of methotrexate occurs during the flow of 0.04 M phosphate buffer (pH 3.5) containing plasma sample through CTH column with a flow rate of 0.5 mL/min at 40 degrees C. The fluorescent products were transferred to the protein-coated precolumn, which was then flushed with the same buffer for clean-up and enrichment from plasma sample. The trapped substances were desorbed from the precolumn and eluted onto the ODS/TM analytical column by isocratical elution with a mobile phase containing 0.05 M phosphate buffer, pH 6.6 and acetonitrile (90-10, v/v) for subsequent separation. The fluorescent products were detected fluorimetrically at excitation and emission wavelengths of 367 and 463 nm, respectively. The complete analysis was achieved within 15 min per sample. The calibration graph was linear in the range of 50-500 ng/mL of methotrexate and there was no interference from endogenous components.     

Elevation of soluble IL-2 receptor and IL-4, and nonelevation of IFN-gamma in sera from patients with allergic asthma

This study is the first demonstration of glial fibrillary acidic protein (GFAP)-immunoreactivity in the retina of the lamprey Lampetra fluviatilis. This immunoreactivity is expressed on one hand, in radial processes and somata which belong to Müller cells and, on the other hand, in horizontal fibers in the intermediate plexus between horizontal cells. The tracing of these fibers to Müller cells or horizontal cells is discussed.     

Endothelin-1 levels in induced sputum samples from asthmatic and normal subjects

BACKGROUND: Endothelin-1 (ET-1) is a potent bronchoconstrictor which may have a role in the pathogenesis of asthma. The levels of ET-1 in saliva, induced sputum, and plasma from asthmatic and non-asthmatic subjects were compared. METHODS: Sputum induction was performed on 28 asthmatic subjects and nine normal volunteers. ET-1 levels were measured in plasma, saliva, and sputum samples and reversed phase high performance liquid chromatography (RP-HPLC) was performed on saliva and sputum samples. RESULTS: ET-1 was present in the following order of concentration in both normal and asthmatic subjects: saliva > sputum > plasma (saliva, median 30.1 and 23.9 pg/ ml, respectively; sputum, median 15.5 and 11.2 pg/ml; plasma, median 3.1 and 3.6 pg/ ml). There were no differences between asthmatic and normal subjects in the levels of ET-1 in each fluid. The levels of ET-1 in asthmatic subjects were not influenced by whether or not they were taking inhaled steroids. RP-HPLC of sputum and saliva confirmed the presence of ET-1 in these fluids. CONCLUSIONS: Levels of ET-1 can be measured in saliva and sputum obtained by sputum induction in asthmatic and healthy subjects and, although no difference was found in basal levels of ET-1 in sputum, saliva and plasma between normal subjects and asthmatics without bronchoconstriction, it is apparent that ET-1 is produced or released locally within the respiratory tract in concentrations higher than those in plasma.     

Persistence of sputum eosinophilia in children with controlled asthma when compared with healthy children

We aimed to describe induced sputum cell counts in healthy nonasthmatic children, and to compare these to children with controlled and uncontrolled asthma. Following clinical assessment and spirometry, ultrasonically nebulized hypertonic saline was used to induce sputum from children with asthma (n=50) and without asthma (n=72). Sputum was dispersed and cell counts performed to yield total and differential cell counts. Specific stains were used for eosinophil and mast cell counts. All of the children with asthma were receiving inhaled and/or oral corticosteroids. Current asthma control was assessed in terms of symptoms and lung function. Children were classified as controlled on inhaled corticosteroids (no current symptoms, normal lung function n=15), current symptomatic asthma (n=16) and asthma exacerbation (n=11). It was found that eosinophils comprised a median 0.3% (interquartile range (IQR): 0, 1.05) of cells in sputum from healthy children. Sputum eosinophils (4.3% (IQR: 15, 14.1) p=0.0005) and epithelial cells (14% (IQR: 6, 19.4) p=0.0005) were significantly higher in children with asthma than in nonasthmatic children. Children whose asthma was controlled, as well as those with symptoms, had more sputum eosinophils and epithelial cells than the nonasthmatics. Mast cells were found in the sputum of only four of the 42 children with asthma. This study demonstrates that eosinophilic airway inflammation and epithelial damage can occur in children with asthma. Airway inflammation persists even in those children who are receiving inhaled corticosteroids, have normal lung function and good symptomatic control of their disease.     

氟替卡松/沙美特罗对哮喘患者外周血单个核细胞中辅助性T细胞亚群及气道炎症的影响

目的:探讨氟替卡松/沙美特罗对哮喘患者外周血单个核细胞中辅助性T细胞亚群(Th)及气道炎症的影响,为其临床治疗哮喘提供理论依据.方法:13例慢性持续期中度哮喘患者,给予氟替卡松/沙美特罗连续治疗1.5和3.0个月.检测治疗前后外周血Th亚群(Th1、Th2和Th3)、血清细胞因子及IgE水平,观察诱导痰炎症细胞的变化和临床疗效.结果:氟替卡松/沙美特罗治疗1.5个月时,与治疗前比较,哮喘患者外周血Th3百分数增加、Th2百分数减少(P<0.05),转化生长因子β(TGF-β)、白细胞介素4(IL-4)无明显变化(P>0.05);痰嗜酸细胞(eos)百分比明显降低(P<0.01),血eos及痰中性粒细胞百分比无明显变化;呼气峰流速值 (PEF)、哮喘控制测试(ACT)评分明显增加,PEF变异率明显降低(P<0.01),每日急救药物使用无明显减少.治疗3个月时,与治疗前比较,Th1、Th1/Th2、Th3明显增加,Th2明显减少(P<0.05或P<0.01);IL-4、TGF-β明显降低(P<0.05),干扰素γ(IFN-γ)明显增加(P<0.05);IgE含量无明显变化(P>0.05);血eos、痰eos及痰中性粒细胞均明显降低(P<0.01);PEF、ACT评分明显增加,PEF变异率明显降低(P<0.01),每日急救药物使用次数明显减少(P<0.01).治疗3.0个月与治疗1.5个月时比较,PEF变异率继续降低、ACT评分继续增加(P<0.01).结论:氟替卡松/沙美特罗能提高哮喘患者外周血Th3数量,纠正Th1/Th2失衡,减轻气道炎症和临床症状,且随治疗时间延长,效果进一步提高.     

Value of assessing cryptococcal antigen in bronchoalveolar lavage and sputum specimens from patients with AIDS

Cryptococcus neoformans has become a significant opportunistic pathogen, accounting for 8-10% of infectious complications in patients with AIDS. When encapsulated yeast cells are observed in Giemsa-stained smears of bronchoalveolar washings (BAL), or induced sputum specimens, confirmation as C. neoformans is germane to definitive therapy. We therefore studied 30 BAL and 9 induced sputum specimens for cryptococcal antigen. Of the 30 BAL, 3 specimens were positive for cryptococcal antigen, ranging in titer from 1:4 to 1:256, and 2 of 9 sputum samples were also smear, culture and antigen positive (titer 1:2) for C. neoformans. Of the 34 negative specimens, none of the seven containing Candida species or the one containing H. capsulatum or the one containing P. carinii cross-reacted with cryptococcal anticapsular antibody. Our results indicate that when yeast forms suggestive of C. neoformans are visualized on direct smears of BAL or sputum samples, rapid confirmation as C. neoformans may be achieved by assessment for cryptococcal antigen. A correlation may also exist between antigen titer in respiratory specimens and extent of cryptococcal infection.     

Non-invasive monitoring of bronchial inflammation in asthma

The present consensus on the diagnosis and treatment of asthma relies on symptoms and lung function measurements for the monitoring of disease severity. Even though this probably remains the cornerstone of asthma management, the rapidly increasing insight into the pathogenesis and pathophysiology of the disease is presently leading to the development of more direct measurements of airway inflammation, which may provide potentially relevant information on its clinical course and prognosis. However, at present none of these has sufficiently been validated for current use in monitoring patients with asthma. First, there are new ways of looking at symptoms and lung function. Careful measurements of symptoms by visual analogue scale (VAS) are suggesting that inflammatory activity within the airways can be subjectively perceived, a sensitivity which may be blunted in patients with brittle asthma. In addition, modern physiological parameters, such as the degree of bronchodilatation following a deep breath (M/P-ratio), are strongly associated with airway inflammation. Second, there are multiple cellular and/or soluble markers of inflammation in peripheral blood (using PCR, in situ hybridization, flow cytometry, or circulating mediators and cytokines) and in urine (LTE4, EPX). Recently this has been extended by similar measurements in hypertonic saline-induced sputum (cell differentials and specific stainings on cytospins, flow cytometry, and levels of e.g. ECP, IL-5, IL-8). Finally, mediators and cytokines in the condensate of exhaled air (H2O2, leukotrienes, IL-5?) as well as exhaled NO are currently under evaluation. Adding such markers of airway inflammation as guides in asthma therapy is potentially useful. As a first step towards such a new approach we have recently shown that adding the reduction of airway hyperresponsiveness to the aims of asthma therapy leads to a better clinical as well as histological outcome after two years of treatment. In conclusion, there are new and exciting perspectives in the monitoring of disease severity in asthma in the future. Longitudinal studies presently ongoing will elucidate which parameter is potentially most useful in guiding asthma management.     

Elevation of soluble CD23 in sera from patients with infectious mononucleosis

CD23 is induced in B cells upon infection by Epstein-Barr virus (EBV) and a soluble form (soluble CD23: sCD23) is found in culture supernatants from EBV-transformed B cell lines. Based on these observations, we measured serum sCD23 levels in patients with infectious mononucleosis (IM) caused by EBV infection. Sera from patients with IM at the time of diagnosis contained more sCD23 than sera from normal control subjects. Changes in serum sCD23 levels during the course of disease showed that serum sCD23 levels were elevated at the time of diagnosis and they decreased to the normal levels during the convalescent phase defined by the improvement of symptoms of IM. These results indicate that the elevated levels of sCD23 were observed at the acute phase of IM and may be useful in diagnosing IM.     

Increased iron and ferritin content of sputum from patients with cystic fibrosis or chronic bronchitis

PURPOSE: Extracellular free iron, or iron bound to ferritin, may promote oxidative injury and bacterial growth in airways of patients with chronic airway inflammation due to cystic fibrosis (CF) or chronic bronchitis (CB). In this study, we assessed sputum content of total iron, ferritin, and transferrin in patients with CF or CB as well as sputum from normal subjects with acute airway inflammation caused by viral upper respiratory tract infections (URTIs). METHODS: Spontaneously produced sputum was obtained from 33 subjects, including 10 subjects with CF, 18 subjects with CB (10 acute exacerbations, 8 with stable CB), and 5 subjects with URTIs (control subjects). After lysing and dilution, total iron concentrations were determined by controlled coulometry, ferritin was measured by radioimmunoassay, and transferrin was measured by enzyme-linked immunosorbent assay. RESULTS: Iron was not present in detectable amounts in control sputums, but ferritin was present (6+/-2 ng/mg protein, mean+/-SE), as was transferrin (2.37+/-0.44 microg/mg). Compared with control subjects, concentrations of iron in sputum were increased in patient groups with higher amounts in CF patients (242+/-47 ng/mg, p<0.01) than CB patients with acute exacerbations or patients with stable CB (98+/-50 and 42+/-12 ng/mg, p<0.05 for both). Ferritin content of sputum was also increased in each group, with CF patients (113+/-22 ng/mg, p<0.001) higher than CB patients (acute, 45+/-10 ng/mg; stable, 87+/-24 ng/mg; p<0.01 for both). Compared with control subjects, sputum transferrin was decreased in CF patients (1.09+/-0.40 microg/mg, p<0.05), but not CB patients. CONCLUSIONS: These findings indicate there are increased airway concentrations of total iron and ferritin-bound iron in patients with CB and, to a greater extent, in patients with CF. Particularly in CF patients who also demonstrated decreased airway concentrations of transferrin, ferritin-bound iron in airways may promote oxidative injury and enhance bacterial growth.