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A novel flow cytometric-based method to measure kinase inhibition in sputum from COPD subjects

Title: A novel flow cytometric-based method to measure kinase inhibition in sputum from COPD subjects
Author(s): Nicholson, GC
Holloway, RA
Leaker, BR
Kilty, I
Salganik, M
Tan, L
Barnes, PJ
Donnelly, LE
Item Type: Journal Article
Abstract: Janus kinases (JAKs) regulate inflammatory gene expression through phosphorylation of signal transducer and activator of transcription (STAT) proteins. Expression of STAT proteins is increased in chronic obstructive pulmonary disease (COPD), and may be involved in driving chronic inflammation. Oral JAK inhibitors are effective as anti-inflammatory therapy but exhibit dose-limiting adverse effects. Development of inhaled compounds would be enhanced by robust biomarkers that directly reflect the anti-inflammatory and pharmacological activity in the lung.A novel flow cytometry assay was developed to measure STAT1 phosphorylation in sputum inflammatory cells. The standard sputum processing method was refined to improve sputum cell viability. The flow cytometric assay was used to assess the reproducibility of the measurement of STAT1 phosphorylation and the in vitro activity of a pan JAK-inhibitor on three separate visits in patients with COPD.Upregulation of STAT1 phosphorylation was measured following in vitro IFNγ stimulation of sputum macrophages (stimulated/unstimulated ratio 1.57; p<0.00001). Upregulation was inhibited following in vitro preincubation with a pan JAK-inhibitor (inhibited+stimulated/unstimulated ratio 0.97). STAT1 phosphorylation activity could only be measured in macrophages.Sputum from patients with COPD can be used to reproducibly measure phospho-STAT expression in sputum macrophages. The flow cytometry-based method can be used to evaluate kinase inhibitors in vitro and subsequently in ex vivo studies. The assay is particularly useful for the assessment of inhaled compounds where whole blood assays may not be relevant.
Publication Date: 22-Jun-2016
Date of Acceptance: 3-Jun-2016
URI: http://hdl.handle.net/10044/1/37083
DOI: http://dx.doi.org/10.1136/bmjresp-2016-000140
ISSN: 2052-4439
Publisher: BMJ Publishing Group
Journal / Book Title: BMJ Open Respiratory Research
Volume: 3
Issue: 1
Copyright Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Keywords: COPD Pharmacology
Cytokine Biology
Macrophage Biology
Publication Status: Published
Article Number: e000140
Appears in Collections:National Heart and Lung Institute
Airway Disease
Faculty of Medicine



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