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Bronchodilator reversibility (BDR) was expressed as per cent FEV1 change compared to baseline value. The study was approved by the Ethical Committee of our Institution, and written consent was obtained from children��s parents. Fiberoptic bronchoscopy was performed as previously described (12). BAL was carried out using normal sterile saline solution warmed to body temperature (37��C). BAL volume Pomalidomide was adjusted to body weight using 1?ml/kg divided into three equal fractions in children weighing 20?kg (29). The fluid was recovered by mechanical aspiration into a suction trap using pressure of 25�C100?mmHg. The first sample collected was used for culture, and values >105?CFU/ml of BAL fluid were considered to indicate bacterial infection (12). The other two aliquots were pooled together and submitted for cytological studies and analysis of BAL components, buy Nutlin-3a including proteins and inflammatory mediators. BAL was considered technically acceptable if recovery was ��40%. Furthermore, in 59 out of the 191 children included in the study, bronchial biopsies were available. These have been evaluated to quantify the eosinophil infiltration in other studies and processed as previously described (20, 21, 24). The data have been used to investigate correlations between tissue eosinophilia and the parameters examined in the present work. Before assessing the cellular and noncellular component, the fluid was filtered through gauze and centrifuged at 500?g for 10?min. After eliminating the supernatant, the cell pellet was suspended in 1?ml of Hank��s balanced salt solution (Ca2+ and Mg2+ free) and centrifuged (Phicom Co., Chescire, UK), spinning?= 1?��?106?cells/ml. The centrifuged cell preparations were air-dried and stained with May-Grumwald-Giemsa for the differential count of macrophages, lymphocytes, eosinophils and neutrophils. The total cell values and the different cell populations were expressed as 106/ml and in per cent of total cell count. The quantitative evaluation of BAL ECP was performed using an immunofluorescence assay (ImmunoCAP 250 and ImmunoCAP 1000; Phadia, Milan, Italy). The lower limit of detection for ECP in BAL was 2?��g/l, and the upper limit was 200?��g/l. IL-8 and TNF�� analyses were performed using Oxymatrine the Immulite assay (Immulite one; Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA). The technique is based on a solid-phase two-site chemiluminescent enzyme immunometric assay. The lower limit of detection for IL-8 was 1?pg/ml, and the upper limit was 7500?pg/ml. The lower limit of detection for TNF�� was 1.7?pg/ml, and the upper limit was 1000?pg/ml. Eotaxin measurement was performed by a quantitative sandwich enzyme immuno-assay (R&D Systems, Minneapolis, MN, USA). Results were expressed in pg/ml, and minimum detectable dose is
