Fei Wang1, Bei He. 1. Respiratory Department, Third Hospital, Peking University, Beijing 100083, China.
Abstract
BACKGROUND: Induced sputum analysis has been widely used to assay airway inflammation. However, the mucolytic agent dithiothreitol (DTT) may affect the detection of some soluble mediators. OBJECTIVES: Our study was designed to investigate whether DTT affects the detection of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha and interleukin (IL)-10. METHODS: Sputum samples were collected from 12 patients with chronic obstructive pulmonary disease (COPD) who were current or ex-smokers. The patients inhaled a 3% saline solution for 30 min from an ultrasonic nebulizer. Half of the sample collected was treated with 0.1% DTT and the other half was homogenized with phosphate-buffered saline (1x PBS) at room temperature. The total cell counts of the two portions were compared and the concentrations of MCP-1, MIP-1alpha and IL-10 in the supernatant were measured using immunoassays. RESULTS: The median ratio of the forced expiratory volume in 1 s (FEV(1)) to the forced vital capacity was 46.0% (interquartile range 43.0-58.0%), and the median predicted FEV(1)% was 41% (interquartile range 36.0-44.0%). Sputum treated with DTT had higher total cell counts (p < 0.01) but lower levels of MCP-1 and MIP-1alpha (p < 0.02, p < 0.05) than that treated with PBS. The concentration of IL-10 was similar in DTT-treated and PBS-treated samples. CONCLUSIONS: Sputum samples treated with DTT were homogenized more effectively and cell recovery was greater than in those treated with PBS. However, the detectable concentrations of MCP-1 and MIP-1alpha were significantly reduced in the sputum of DTT-treated samples.
BACKGROUND: Induced sputum analysis has been widely used to assay airway inflammation. However, the mucolytic agent dithiothreitol (DTT) may affect the detection of some soluble mediators. OBJECTIVES: Our study was designed to investigate whether DTT affects the detection of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha and interleukin (IL)-10. METHODS: Sputum samples were collected from 12 patients with chronic obstructive pulmonary disease (COPD) who were current or ex-smokers. The patients inhaled a 3% saline solution for 30 min from an ultrasonic nebulizer. Half of the sample collected was treated with 0.1% DTT and the other half was homogenized with phosphate-buffered saline (1x PBS) at room temperature. The total cell counts of the two portions were compared and the concentrations of MCP-1, MIP-1alpha and IL-10 in the supernatant were measured using immunoassays. RESULTS: The median ratio of the forced expiratory volume in 1 s (FEV(1)) to the forced vital capacity was 46.0% (interquartile range 43.0-58.0%), and the median predicted FEV(1)% was 41% (interquartile range 36.0-44.0%). Sputum treated with DTT had higher total cell counts (p < 0.01) but lower levels of MCP-1 and MIP-1alpha (p < 0.02, p < 0.05) than that treated with PBS. The concentration of IL-10 was similar in DTT-treated and PBS-treated samples. CONCLUSIONS: Sputum samples treated with DTT were homogenized more effectively and cell recovery was greater than in those treated with PBS. However, the detectable concentrations of MCP-1 and MIP-1alpha were significantly reduced in the sputum of DTT-treated samples.
Authors: Helena M Linge; Cecilia Andersson; Sara L Nordin; Anders I Olin; Ann-Cathrine Petersson; Matthias Mörgelin; Amanda Welin; Johan Bylund; Leif Bjermer; Jonas Erjefält; Arne Egesten Journal: Mol Med Date: 2013-09-30 Impact factor: 6.354
Authors: Andrew Higham; Paul Cadden; Thomas Southworth; Matthew Rossall; Umme Kolsum; Simon Lea; Richard Knowles; Dave Singh Journal: ERJ Open Res Date: 2016-10-26