Systemic corticosteroids have been recommended as a therapeutic option in patients with moderate to severe COPD. In an early stage of the disease, i.e. chronic bronchitis with mild or no airflow obstruction, a trial with inhaled steroids could reveal potential benefits, particularly in terms of a modulation of airway inflammation. We therefore investigated the effect of inhaled fluticasone (1000 microg day(-1)) on markers of airway inflammation in 19 patients with chronic bronchitis (mean+/-SEM FEV1, 83.4+/-3.0% predicted; FEV1/VC, 67.5+/-2.4%) in a double-blind, cross-over, placebo-controlled manner. Visits were performed before and after two 4-week treatment periods. separated by a 4-week washout period. Lung function, the concentration of exhaled nitric oxide, differential cell counts in induced sputum and the number of cells positive for iNOS, as well as the levels of LDH, ECP, neutrophil elastase and IL-8 in sputum supernatants were determined. Although the total cell number decreased significantly after fluticasone (geometric mean 12.3 vs. 7.7 x 10(6)/ml; P<0.05) it was not significantly different from the change observed after placebo (14.2 vs. 10.6 x 10(6)/ml; n.s.). None of the other parameters showed statistically significant changes after fluticasone or placebo and the results did not depend on the presence of airway hyperresponsiveness. We conclude that in patients with chronic bronchitis short-term treatment with inhaled corticosterids did not improve lung function or inflammatory parameters to an extent which was statistically significant as compared to spontaneous variability.

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Pretreatment with dithiothreitol (DTT) is necessary to dissolve mucus in samples of induced sputum prior to analysis. However, DTT may affect cell surface markers which are essential for lymphocyte subtyping. Therefore, the aim of this study was to evaluate the effect of DTT on an appropriate panel of surface markers. Peripheral blood leukocytes were used because these cells, in contrast to sputum cells, could be obtained without DTT treatment.Peripheral blood from healthy donors was incubated with either DTT according to standard sputum procedures or phosphate-buffered saline (PBS), washed and incubated with fluorochrome-labelled antibodies. After lysis of erythrocytes, analysis was performed using a calibrated flow cytometer. Leukocyte populations were identified by their light scattering properties. For analysis, fluorescence intensity was compared between DTT-and PBS-treated samples.After treatment with DTT, fluorescence intensity was significantly increased in CD16-positive granulocytes; it was reduced in CD2-positive lymphocytes, CD45-positive lymphocytes and CD14-positive monocytes (p#0.001). These changes occurred in all samples. The fluorescence intensity of CD3-, CD4-, CD8-, CD19-, CD56-and histocompatibility leukocyte antigen DR-positive lymphocytes was not altered by DTT. However, there were statistically significant (p<0.001), although small, changes in the percentages of leukocytes.The present data demonstrate that, although dithiothreitol as used in sputum analysis affects some surface markers of peripheral blood leukocytes, comparability between samples concerning lymphocyte surface markers is preserved. Therefore, it is suggested that treatment of sputum samples with dithiothreitol does not invalidate the immunocytochemical analysis of lymphocytes. Eur Respir J 2000; 16: 324±329. The method of induced sputum is widely used as a noninvasive procedure for obtaining biological samples from the airways [1,2] in order to determine their cellular and biochemical composition in relation to airway diseases [3,4]. As sputum cells are embedded in airway secretions, the material has to be liquified in order to prepare singlecell suspensions for flow cytometry. This can be achieved by incubation with the potent reducing agent dithiothreitol (DTT, 2,3-dihydroxybutane-1,4-dithiol) [5]. Therefore, DTT is widely used in sputum processing [6±9]. However, owing to its reducing properties, DTT could affect the three-dimensional structure of proteins, which is maintained by disulphide bonds, thereby leading to changes in the availability and integrity of epitopes which could hamper immunological detection procedures [10].Indeed, the data which are available indicate effects of DTT or dithioerythritol (DTE) on surface markers assessed by flow cytometry on eosinophils or neutrophils even if cell viability remains unchanged [11±13]. Data regarding markers that are essential for lymphocyte subtyping have been published in abstract form [14]. Recent data have shown improved cell and sputum supernatant inflamma...

Neutrophil-dominated endobronchial inflammation is a major characteristic of cystic fibrosis (CF) and there is increasing demand for easy-to-perform noninvasive monitoring for prediction and intervention.Fourteen stable paediatric CF patients (8-17 yrs; mean forced expiratory volume in one second 86.7% of the predicted value) were investigated once by fractional bronchoalveolar lavage (BAL) and by sputum induction on three occasions, 2-6 weeks apart. Sputum was induced by consecutive 10-min inhalations of 3, 4 and 5% saline. CF sputum cellular profiles were compared with BAL fluid cell counts and samples from agematched healthy children, and between different time points to assess reproducibility.Adequate sputum was recovered on w95% of occasions. In all sputum fractions, CF patients showed higher neutrophil counts than healthy children. Neutrophil percentages were highest in the first BAL fraction (median 92%), followed by sputum, in which the percentages decreased in consecutive fractions (72, 66 and 64%), whereas counts were lowest in the pooled BAL fraction (53%). Increasing percentages of macrophages mirrored the decreases in neutrophil percentage. Results of sputum induction at different time points in the CF patients showed good reproducibility and nonoverlap with counts from healthy children.In conclusion, the results of sputum induction in children with mild stable cystic fibrosis adequately describe airway inflammation by providing cellular profiles with lower relative neutrophil counts than in the first ("bronchial") bronchoalveolar lavage fraction and higher relative neutrophil counts than in subsequent pooled ("more peripheral") bronchoalveolar lavage fractions. Eur Respir J 2003; 22: 497-502.

Our results indicate that the freezing of sputum samples at different stages of processing does not alter sputum morphology to an extent that affects the results of differential cell counts.