Strategy Aimed at Reduction of Sputum Eosinophils Decreases Exacerbation Rate in Patients with Asthma

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1 The Journal of International Medical Research 2006; 34: Strategy Aimed at Reduction of Sputum Eosinophils Decreases Exacerbation Rate in Patients with Asthma J CHLUMSKÝ 1, I STRIZ 2, M TERL 3 AND J VONDRACEK 4 1 Department of Pneumology, 1st Medical Faculty, Charles University and Thomayer Faculty Hospital, Prague, Czech Republic; 2 Department of Immunology, Institute for Clinical and Experimental Medicine, Prague, Czech Republic; 3 Department of Pulmonary Diseases and Tuberculosis, Faculty Hospital Plzen, Czech Republic; 4 Institute of Mathematics, Czech Academy of Sciences, Prague, Czech Republic Under Global Initiative for Asthma guidelines, the clinical control of disease activity and the adjustment of treatment in patients with asthma are based on symptoms, use of rescue medication, lung function and peak expiratory flow measurement (standard strategy). We investigated whether a strategy to reduce the number of sputum eosinophils (EOS strategy) gives better clinical control and a lower exacerbation rate compared with the standard strategy. Fifty-five patients with moderate to severe asthma entered this open, randomized, parallel-group study and visited the out-patient department every 3 months for 18 months. The dose of corticosteroids was adjusted according to the standard strategy or the percentage of sputum eosinophils (EOS strategy). During the study period, the EOS strategy led to a significantly lower incidence of asthma exacerbations compared with the standard strategy group (0.22 and 0.78 exacerbations per year per patient, respectively). There were significant differences between the strategies in time to first exacerbation. KEY WORDS: CORTICOSTEROIDS; ASTHMA; INDUCED SPUTUM; EOSINOPHILS; CLINICAL CONTROL Introduction Asthma is a chronic inflammatory disorder characterized by recurrent episodes of breathlessness and wheezing that are accompanied by widespread narrowing of the airways. 1 Chronic inflammation, characterized by infiltration of the airways with inflammatory cells such as eosinophils, secretion of proinflammatory mediators, increased vascular permeability, and mucus production, is believed to be the underlying cause of asthma and an important determinant of its severity. 1 Adequate therapy of asthma should therefore be directed at suppressing airway inflammation. However, there is only a weak relationship between the degree of airway inflammation and the clinical markers, such as lung function and symptoms of asthma, that are currently recommended for the assessment of asthma severity and the adjustment of antiinflammatory treatment. 2,3 Recently, much interest has been focused on the eosinophil count in induced sputum as a minimally invasive, reliable, valid and responsive marker for monitoring airway 129

2 inflammation. 4,5 It has been shown that number of sputum eosinophils is elevated in asthma 5 and decreases after the inhalation of corticosteroids. 6,7 The eosinophil count in induced sputum has also been reported to predict the exacerbation of asthma after steroid withdrawal. 8 Recently, Green et al. 9 showed that a treatment strategy directed at suppressing eosinophilic inflammation in the airways leads to a significant reduction in asthma exacerbation compared with the strategy based on the British Thoracic Society guidelines. 10 The aim of our study was to compare the standard strategy of asthma severity assessment with a strategy based on reducing the number of sputum eosinophils in terms of the incidence of asthma exacerbations, changes in lung function, the frequency of symptoms, and the final dose of inhaled corticosteroids. We expected that the strategy based on reducing the number of sputum eosinophils might improve clinical control and decrease the frequency of exacerbations of asthma in a group of patients with moderate-to-severe asthma. Compared with the study of Green et al., 9 in our study the patients were studied for a longer period of time and different cut-off values for sputum eosinophils were used. Subjects and methods SUBJECTS Subjects with persistent moderate-to-severe asthma that was not controlled on current treatment (forced expiratory volume in 1 s [FEV 1 ] % of predicted; daily dose of inhaled corticosteroid µg of budesonide or equivalent; 11 of the patients were on systemic corticosteroids) visiting an out-patient department were recruited for participation in the study (Table 1). All patients had a history of recurrent dyspnoea, cough and chest tightness and had been treated with inhaled corticosteroids for at least 1 year. The diagnosis of asthma was confirmed by reversible airway obstruction, with FEV 1 of 70% of predicted and a subsequent increase in FEV 1 of 15% after inhalation of 200 µg of salbutamol using a metered dose inhaler connected to a spacer device, and/or diurnal peak expiratory flow (PEF) variation of 20% or more on at least 4 of 14 days of a run-in period. All patients were atopic, as assessed by a positive skinprick test (Stallergenes, Antony, France; weal of > 3 mm) to one or more common aeroallergens. None of the patients had a history of respiratory disease other than asthma and none had a history of upper respiratory tract infection within the month preceding the study. Current smokers were not included in the study. The study was approved by the local ethics committee and all patients gave written informed consent. STUDY DESIGN This study was an open, prospective, randomized, parallel-group trial over a period of 18 months. After a 2-week run-in period, patients were randomized into two groups with different strategies of disease monitoring. A treatment strategy designed to reduce the number of eosinophils in induced sputum (EOS strategy) was compared with the standard strategy recommended by international guidelines. Randomization was done by an independent investigator (JV) using a computer program and was stratified by the dose of inhaled steroids, treatment with systemic steroids and add-on therapy with inhaled long-acting β 2 agonists and theophyllines. The patients attended the out-patient department in the morning at intervals of 3 months during the 18 months of the study period. All patients were asked to record their symptoms and medication usage and were instructed to perform PEF 130

3 TABLE 1: Demographic and baseline characteristics of asthma patients managed with a strategy based on symptoms, medication usage and lung function (standard strategy) and patients managed with a strategy based on sputum eosinophil count (EOS strategy) Standard strategy EOS strategy Difference Number NA Gender (male/female) 6/15 13/17 NS Age (years) 48 ± ± 19 NS History of asthma (years) 11 ± 7 10 ± 9 NS Exacerbation rate during the year before randomization NS Inhaled corticosteroid (µg/day) 1418 ± ± 1043 NS Systemic corticosteroid (mg/day) 3 ± 8 4 ± 9 NS Proportion of patients on add-on therapy NS FEV 1 (% predicted) 71.3 ± ± 20 NS Post-FEV 1 (% predicted) 75.1 ± ± 19.7 NS PEF (% predicted) 74.8 ± ± 19.7 NS FEV 1 /VC (%) 67.9 ± ± 11.2 NS Frequency of daytime symptoms per week 17.6 ± ± 15.2 NS Frequency of night-time symptoms per week 3.1 ± ± 2.3 NS Frequency of rescue medication per week 13.8 ± ± 36.9 NS Data are mean ± SD. Dose of inhaled corticosteroid is expressed as µg/day of budesonide; dose of systemic corticosteroid is expressed as mg/day of methylprednisolone. FEV 1, forced expiratory volume in 1 s; post-fev 1, FEV 1 15 min after inhalation of 200 µg salbutamol via metered dose inhaler; PEF, peak expiratory flow; FEV 1 /VC, ratio of FEV 1 to vital capacity; % predicted, percentage of predicted value; NA, not applicable; NS, not significant. measurements twice a day, in the morning and in the evening. At each visit a physical examination and pre- and postbronchodilator spirometry were performed. Additionally, the patients randomized to the EOS strategy arm underwent sputum induction. In the standard strategy arm, the dosage of inhaled or systemic corticosteroid was adjusted at the end of each study visit by the treating physician (JC, MT) using the standardized algorithm shown in Table 2, while in the EOS strategy arm management decisions were made by an independent physician (IS), who was blinded to the patients clinical data, within 1 week after each study visit, by a telephone call. Treatment with inhaled corticosteroid was adjusted in a stepwise manner (Table 2) with three possible steps at each time-point: (i) no change; (ii) halving the dose of corticosteroids (step down); and (iii) doubling the dose of corticosteroids (step up). 131

4 Alternatively, when the daily dose of inhaled corticosteroids reached 3200 µg of budesonide or equivalent, 16 mg/day of methyl-prednisolone was instituted, with gradual tapering within 1 month. In the standard strategy group, the adjustments were guided by information about the frequency of symptoms and the use of rescue salbutamol, morning PEF values and PEF variation. In the EOS strategy group, adjustments depended on the percentage of sputum eosinophils, and were intended to maintain the count below 8% (Table 2). The limit of 8% reflects the results of studies showing that exacerbations rarely occur in patients with fewer than 10% of eosinophils in induced sputum. 8,11,12 The marker that indicated the greatest loss of asthma control was always used. To reflect international recommendations and current clinical practice, concomitant medication with theophyllines and longacting β 2 agonists was allowed when appropriate, provided that their doses remained constant during the study and the proportion of patients receiving add-on therapy was similar in the two groups. CLINICAL METHODS Pre- and post-bronchodilator flow-volume loops were measured using the Zan 100 computerized spirometric system with a pneumotachograph (Flowhandy II ; Oberthulba, Germany) and the best of three attempts not varying by more than 5% was used for analysis. 13 A reversibility test was performed after inhalation of 200 µg of salbutamol via a metered dose inhaler connected to a spacer device, and postbronchodilator values were obtained within 15 min after administration of salbutamol. Long-acting β 2 agonists and theophyllines were withheld for at least 14 h and no shortacting inhaled bronchodilator was allowed within 6 h before measurements were made. The frequencies of daytime and nighttime asthma symptoms (dyspnoea, cough and chest tightness) and the number of rescue salbutamol inhalations were recorded in a diary card daily. Electronic peak flow meters (Amos-1 ; Jaeger-Toennies, Höchberg, Germany) storing up to 400 measurements were used for PEF measurements. Data from the Amos-1 meters were transferred to and analysed using an TABLE 2: Criteria used for the adjustment of corticosteroid doses in asthma patients managed with a strategy based on symptoms, medication usage and lung function (standard strategy) and patients managed with a strategy based on sputum eosinophil count (EOS strategy) Standard strategy Frequency Frequency EOS of daytime of night-time strategy Morning symptoms or symptoms or Sputum PEF/variation SABA/week SABA/week eosinophils Step down > 80%/< 15% < 4 < 1 3 No change 60 80%/< 20% Step up < 60%/> 20% > 7 > 2 8 PEF, mean peak expiratory flow expressed as percentage of personal best values; variation, diurnal variation in PEF; SABA, rescue short-acting inhaled salbutamol. Sputum eosinophils are expressed as percentage of total cell count. 132

5 original computer program at each visit. Peak flow variation was calculated from the difference between the highest and the lowest daily readings divided by the highest reading multiplied by 100. Frequency of symptoms, number of puffs of rescue salbutamol and PEF measurements over the last 14 days before each study visit were used for analyses and treatment adjustments. OUTCOME VARIABLES The rate of asthma exacerbations was the main outcome variable. For the purpose of this study, an exacerbation was defined as a doubling of the frequency of symptoms and/or number of puffs of rescue salbutamol and/or a reduction in morning PEF by 30% or more on at least two consecutive days. A decrease in FEV 1 by 30% or more at any study visit was also considered a sign of asthma exacerbation. The patients were instructed to take 16 mg of methylprednisolone each morning for 10 days if they fulfilled the exacerbation criteria and to call the treating physician (JC or MT) on the occasion of an exacerbation and every other day until resolution. The values of FEV 1, postbronchodilator FEV 1 and FEV 1 /inspiratory vital capacity ratio were the remaining outcome variables. SPUTUM INDUCTION AND PROCESSING Sputum was induced by inhalation of hypertonic saline (3%) at room temperature from an ultrasonic nebulizer (DeVilbiss, Wollaston, UK) at an aerosol output of 4 ml/min. Subjects were encouraged to cough at 2-min intervals until an adequate amount of sputum had been obtained. They were instructed to wash their mouth before each cough. Sputum samples were collected into sterile containers and processed within 2 h. The flow-volume loop measurement was repeated after each sputum induction. The volume of the whole sputum sample was measured and an equal amount of 1% dithiothreitol (Sigma Chemicals, Poole, UK) dissolved in phosphate-buffered saline at 1:10 was added. The mixture was vortexed at room temperature until the sputum had been homogenized, and then filtered through 48-µm nylon gauze. The samples were then centrifuged at 1200 rpm for 10 min and the supernatant was aspirated. The cell pellets were resuspended in phosphate-buffered saline and dispersed on slides, which were subjected to May Grünwald Giemsa staining for differential cell counting. All slides were counted by a technician blinded to the clinical characteristics of the patient. Two slides were used for counting and 300 non-squamous cells were counted on each slide. STATISTICAL ANALYSIS Unpaired Student s t-test was used to test differences in baseline parameters between the two strategy arms. Paired and unpaired Student s t-tests were used to test differences in outcome variables within and between the two strategies. Differences in outcome variables between the strategy arms at the end of the follow-up period were also tested with respect to baseline data by analysis of covariance. The incidence of asthma exacerbations per patient per year was calculated and exacerbations were categorized according to frequency into three groups: (i) no exacerbation; (ii) one exacerbation; (iii) two or more exacerbations. Differences were tested using a χ 2 contingency table. The cumulative incidence of exacerbations was expressed by the Kaplan Meier method, using the first exacerbation only. Values of P < 0.05 were considered significant. All statistical analyses were performed 133

6 with the statistical software SAS System 8.02 (SAS Institute, Cary, North Carolina, USA). Results Of the 55 patients who were enrolled in the run-in period, 25 were randomized to the standard strategy arm and 30 to the EOS strategy arm. Fifty-one patients completed the 18-month follow-up period. Four patients (all in the standard strategy arm) dropped out of the study: two withdrew for protocol violation and two were lost to follow-up. There were no significant differences between the groups with respect to age, gender, atopy, dose of inhaled and systemic steroids, number of patients with add-on therapy, or baseline pre- and post-bronchodilator pulmonary function (Table 1). Despite the fact that hypertonic saline was used for sputum induction, the procedure was well tolerated and the mean fall in FEV 1 was 9.3% (range %). In the EOS strategy arm, the baseline sputum eosinophil count (mean ± SD) was 9 ± 12% and led to increasing the dose of inhaled corticosteroids in 33% and decreasing the dose in 47% of patients at the first visit. During the 18-month study period the eosinophil count in induced sputum fell to 2 ± 2% (change in percentage points 6.1, 95% CI to 1.78; P = ). At baseline and all subsequent visits, a step up was applied in 11.1% and a step down in 29.6% of patients in the standard strategy arm, while in the EOS strategy arm a step up was applied in 21.1% and a step down in 22.2%. In the standard strategy arm, stepping up was indicated in most cases by the frequency of symptoms and/or the number of rescue salbutamol inhalations (94.6%) and in the remaining cases by the PEF recordings (5.4%); the frequency of symptoms and/or the number of rescue salbutamol inhalations prevented decreasing the dose of inhaled or systemic steroids, as indicated by PEF data, in 31.5% of cases. Changes in the dose of inhaled steroids within groups and between the two groups did not reach statistical significance (Fig. 1). The criteria of asthma exacerbation were fulfilled, according to the frequency of symptoms and/or the number of rescue salbutamol inhalations, in 78% of cases in the standard strategy arm and in all cases in the EOS strategy arm. There were significant differences in the number of asthma exacerbations between the two strategy groups. Patients randomized to the standard strategy arm had a higher rate of exacerbations of asthma than those in the EOS strategy arm (0.78 and 0.22 exacerbations per patient per year, respectively; P = 0.013). There were no differences in the number of single exacerbations between the two groups (seven in the standard strategy and six in the EOS strategy arm), but patients in the EOS strategy arm had a lower incidence of two or more exacerbations (two versus seven, P = 0.013). There was a significant difference in the time to first exacerbation between the two strategy arms (χ 2 = 8.8, P = 0.003; Fig. 2). None of the patients had a severe exacerbation requiring hospital admission. The values of FEV 1 as a percentage of those predicted rose during the follow-up period in both groups (change in percentage points 2.7, 95% CI 3.5 to 8.8 in the standard strategy arm; change in percentage points 3.5, 95% CI 2.6 to 9.6 in the EOS strategy arm) but did not reach statistical significance. There were also no significant changes in the post-bronchodilator FEV 1 as a percentage of predicted. The FEV 1 /inspiratory vital capacity ratio decreased in the standard strategy arm (change in percentage points 6.1, 95% CI 10.8 to 1.3) whereas it rose slightly in the EOS strategy arm (change in percentage points 1.2, 95% CI 1.4 to 3.7), 134

7 Dose of IGC (µg/day) Standard strategy EOS strategy P > 0.05 for all time-points Baseline Time (months) FIGURE 1: Change in the doses of inhaled glucocorticosteroids (IGC) during 18 months of follow-up in asthma patients managed with a strategy based on symptoms, medication usage and lung function (standard strategy) and patients managed with a strategy based on sputum eosinophil count (EOS strategy). There were no significant differences between or within groups. Curves represent the mean and bars the SD Patients (%) Standard strategy EOS strategy P = Follow up (months) FIGURE 2: Kaplan Meier plot showing the cumulative incidence of exacerbations in asthma patients managed with a strategy based on symptoms, medication usage and lung function (standard strategy) and patients managed with a strategy based on sputum eosinophil count (EOS strategy). There were significant differences in the time to first exacerbation and the number of patients remaining free of exacerbations. See text for details 135

8 the difference between the two groups being statistically significant (P = 0.009; Fig. 3). Discussion This study has shown that the standard strategy used here, aimed at minimizing symptoms and optimizing lung function, does not necessarily prevent asthma exacerbations in each patient, and therefore the currently recommended guidelines on treatment of asthma seem to be insufficient, at least in a group of patients with moderateto-severe asthma, in optimizing control of the disease. On the contrary, reducing the percentage of sputum eosinophils is more effective in decreasing the frequency of asthma exacerbations in comparison with international guidelines, especially in patients with repeated episodes of exacerbations. Interestingly, according to the percentage of eosinophils in induced sputum, in almost half of the patients the indication was for a step down when adjusting the corticosteroid dose at baseline, and these patients could be assessed as having been overtreated. Although lung function variables are among the physiological and clinical markers currently recommended for the assessment of the control of asthma, their relationship to various invasive and noninvasive indices of airway inflammation is rather weak 14 and does not allow the prediction of a given parameter from another in individual patients. 3,15 Sputum eosinophils are increased in steroid-naive asthma patients and in sensitized subjects after allergen exposure 16 and can be suppressed after variable doses of inhaled or systemic corticosteroids. 17 Thus, sputum examination can identify the presence and intensity of airway inflammation and can be 80 FEV 1 /VC (%) ** 50 Standard strategy EOS strategy Baseline Time (months) FIGURE 3: Changes in the ratio of forced expiratory volume in 1 s (FEV 1 ) to vital capacity (VC) in asthma patients managed with a strategy based on symptoms, medication usage and lung function (standard strategy) and patients managed with a strategy based on sputum eosinophil count (EOS strategy). There was a significant difference between the groups at the end of the study period (**P = 0.009). Curves represent the mean and bars the SD 136

9 used to optimize the dose of corticosteroids needed for adequate suppression of airway inflammation. 18,19 According to this assumption, only a minority of patients in the present study were shown to be using an adequate dose of inhaled or systemic corticosteroid at baseline; most of them required immediate adjustment of their anti-inflammatory treatment. This may have occurred, at least in part, because many of the patients were on add-on therapy with long-acting β 2 agonists or theophyllines, and in these patients symptoms were probably controlled better than their airway inflammation. This supports the hypothesis that combination therapy may enhance the dissociation between markers of clinical control and the degree of airway inflammation. Sputum eosinophils have been shown to have different kinetics of physiological variables after changes in steroid treatment. 11,20 After steroid withdrawal, the increased percentage of sputum eosinophils can herald asthma exacerbation several weeks before the onset of symptoms and worsening of lung function. 11,13 Although there is no clear relationship between increased airway inflammation and exacerbations of asthma, the percentage of sputum eosinophils seems to predict the occurrence of exacerbations. The results of the present study have shown that a strategy aimed at the reduction of sputum eosinophilia leads to a significant decrease in the exacerbation rate and to an improvement in lung function. This finding is in agreement with the results of Green et al., 9 who recently published the first longterm study using sputum eosinophils for the guidance of anti-inflammatory treatment. Although the design of our study differed from that of Green et al., 9 the main outcome variables were similar. Unlike the present study, the study of Green et al. 9 monitored sputum eosinophils in both strategy arms and showed that adjustment of the corticosteroid dose according to signs of clinical control did not lead to adequate suppression of airway inflammation. Our study was designed to compare the clinical efficacies of two different strategies rather than to examine their abilities to suppress airway inflammation. It should be emphasized that exacerbation rates were decreased to similar extents in the two studies. 9 In the present study, the difference in exacerbation rate between the groups was almost four-fold higher than in the study of Green et al., 9 which probably reflects the longer period of follow-up in our study. The results of our study indicate that the threshold level of sputum eosinophils to be maintained in order to decrease the exacerbation rate should be 8% rather than 2.5%, which is considered to be the upper limit of the normal range. In both studies, the mean doses of corticosteroids did not change significantly within the sputum management strategy group, showing that similar proportions of patients were overand undertreated, and the frequencies of step-up and step-down adjustments were almost equal within the two arms. Although the study lacked an appropriate double-blind design, sputum analysis was performed by a blinded technician. Furthermore, the adjustment strategies had strict, predefined limits for changes of corticosteroid doses, which were adhered to accurately. The authors feel that including patients with combination therapy in the present study is somewhat controversial, but the currently recommended guidelines prefer adding long-acting bronchodilators to inhaled corticosteroids rather than doubling their dose, and this study tried to reflect this approach. Moreover, the doses of add-on therapy were kept constant throughout the 137

10 study period to exclude any possible effect of these drugs on the character or degree of airway inflammation. 21 Although longacting β 2 agonists improve symptoms of asthma and lung function, 22 they do not reduce sputum eosinophilia and thus can actually mask the progression of airway inflammation. 23 This may partially explain the large difference in exacerbation rate between the two strategy arms. In conclusion, this study has shown that a treatment strategy aimed at reducing the percentage of eosinophils in induced sputum, in contrast to a strategy using international guidelines, leads to a large decrease in exacerbation rates in patients with moderate-to-severe asthma. The results support the use of sputum eosinophils not only for research purposes but also in clinical practice for the adequate adjustment of corticosteroid doses, at least in patients with asthma that is difficult to control, particularly in those with frequent exacerbations. Acknowledgements This work was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic (Grant No. 5866/3). The authors thank Mrs V. Kubinova for doing the cell counts. Conflicts of interest No conflicts of interest were declared in relation to this article. Received for publication 24 October 2005 Accepted subject to revision 14 November 2005 Revised accepted 21 December 2005 Copyright 2006 Cambridge Medical Publications References 1 National Institutes of Health, NHLBI. Global initiative for asthma. Global strategy for asthma management and prevention. Bethesda, MD. NHLBI/WHO Workshop Report. Publication No , Sont JK, Han J, van Krieken JM, Evertse CE, Hooijer R, Willems LN, et al: Relationship between the inflammatory infiltrate in bronchial biopsy specimens and clinical severity of asthma in patients treated with inhaled steroids. Thorax 1996; 51: Crimi E, Spanevello A, Neri M, Ind PW, Rossi GA, Brusasco V: Dissociation between airway inflammation and airway hyperresponsiveness in allergic asthma. Am J Respir Crit Care Med 1998; 157: Fahy JV, Liu J, Wong H, Boushey HA: Cellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects. Am Rev Respir Dis 1993; 147: Pin I, Gibson PG, Kolendowicz R, Girgis- Gabardo A, Denburg JA, Hargreave FE, et al: Use of induced sputum cell counts to investigate airway inflammation in asthma. Thorax 1992; 47: Lim S, Jatakanon A, John M, Gilbey T, O Connor BJ, Chung KF, et al: Effect of inhaled budesonide on lung function and airway inflammation. Assessment by various inflammatory markers in mild asthma. Am J Respir Crit Care Med 1999; 159: Jatakanon A, Lim S, Chung KF, Barnes PJ: An inhaled steroid improves markers of airway inflammation in patients with mild asthma. Eur Respir J 1998; 12: Jatakanon A, Lim S, Barnes PJ. Changes in sputum eosinophils predict loss of asthma control. Am J Respir Crit Care Med 2000; 161: Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, et al: Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet 2002; 360: British Thoracic Society. The British guidelines on asthma management. Thorax 1997; 52 (Suppl.): S1 S de Kluijver J, Evertse CE, Schrumpf JA, van der Veen H, Zwinderman AH, Hiemstra PS, et al: Asymptomatic worsening of airway inflammation during low-dose allergen exposure in asthma: protection by inhaled steroids. Am J Respir Crit Care Med 2002; 166: in t Veen JC, Smits HH, Hiemstra PS, Zwinderman AE, Sterk PJ, Bel EH: Lung function and sputum characteristics of patients with severe asthma during an induced exacerbation by double-blind steroid withdrawal. Am J Respir Crit Care Med 1999; 160: Quanjer P, Tammeling G, Cotes J, Pedersen OF, Peslin R, Yernault JC: Lung volumes and 138

11 forced ventilatory flows. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J Suppl 1993; 16: Jatakanon A, Lim S, Kharitonov SA, Chung KF, Barnes PJ: Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma. Thorax 1998; 53: Parameswaran K, Pizzichini E, Pizzichini MM, Hussack P, Efthimiadis A, Hargreave FE: Clinical judgement of airway inflammation versus sputum cell counts in patients with asthma. Eur Respir J 2000; 15: Fahy JV, Liu J, Wong H, Boushey HA: Analysis of cellular and biochemical constituents of induced sputum after allergen challenge: a method for studying allergic airway inflammation. J Allergy Clin Immunol 1994; 93: Jatakanon A, Kharitonov S, Lim S, Barnes PJ: Effect of differing doses of inhaled budesonide on markers of airway inflammation in patients with mild asthma. Thorax 1999; 54: Parameswaran K, Pizzichini MM, Li D, Pizzichini E, Jeffery PK, Hargreave FE: Serial sputum cell counts in the management of chronic airflow limitation. Eur Respir J 1998; 11: Claman DM, Boushey HA, Liu J, Wong H, Fahy JV: Analysis of induced sputum to examine the effects of prednisone on airway inflammation in asthmatic subjects. J Allergy Clin Immunol 1994; 94: Pizzichini MM, Pizzichini E, Clelland L, Efthimiadis A, Mahony J, Dolovich J, et al: Sputum in severe exacerbations of asthma: kinetics of inflammatory indices after prednisone treatment. Am J Respir Crit Care Med 1997; 155: Lim S, Tomita K, Caramori G, Jatakanon A, Oliver B, Keller A, et al: Low-dose theophylline reduces eosinophilic inflammation but not exhaled nitric oxide in mild asthma. Am J Respir Crit Care Med 164: Turner MO, Johnston PR, Pizzichini E, Pizzichini MM, Hussack PA, Hargreave FE: Anti-inflammatory effects of salmeterol compared with beclomethasone in eosinophilic mild exacerbations of asthma: a randomized, placebo controlled trial. Can Respir J 1998; 5: Mcivor RA, Pizzichini E, Turner MO, Hussack P, Hargreave FE, Sears MR: Potential masking effects of salmeterol on airway inflammation in asthma. Am J Respir Crit Care Med 1998; 158: Address for correspondence Dr J Chlumský Department of Pneumology, 1st Medical Faculty, Charles University and Thomayer Faculty Hospital, Vídeňská 800, Prague 4, Czech Republic. jenouch@astma.3v.cz 139