Publications
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Nicholson GC, Holloway RA, Leaker BR, et al., 2016, A novel flow cytometric-based method to measure kinase inhibition in sputum from COPD subjects, BMJ Open Respiratory Research, Vol: 3, ISSN: 2052-4439
INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Belchamber KBR, Holloway R, Dunne A, et al., 2015, Comparison Of Budesonide And Fluticasone Propionate On COPD Macrophage And Neutrophil Phagocytosis And Killing Of Bacteria, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Guney TG, Holloway R, Taylor A, et al., 2013, Increased expression of sphingosine-1-phosphate and S1PR2 are associated with attenuated macrophage phagocytosis in COPD, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
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Holloway R, Nicholson G, Leaker B, et al., 2013, Detection of STAT1 phosphorylation in COPD sputum by flow cytometry, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Holloway RA, Donnelly LE, 2013, Immunopathogenesis of chronic obstructive pulmonary disease, CURRENT OPINION IN PULMONARY MEDICINE, Vol: 19, Pages: 95-102, ISSN: 1070-5287
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- Citations: 50
Hackett T-L, Holloway R, Holgate ST, et al., 2008, Dynamics of pro-inflammatory and anti-inflammatory cytokine release during acute inflammation in chronic obstructive pulmonary disease: an ex vivo study., Respir Res, Vol: 9
BACKGROUND: Exacerbations of Chronic obstructive pulmonary disease (COPD) are an important cause of the morbidity and mortality associated with the disease. Strategies to reduce exacerbation frequency are thus urgently required and depend on an understanding of the inflammatory milieu associated with exacerbation episodes. Bacterial colonisation has been shown to be related to the degree of airflow obstruction and increased exacerbation frequency. The aim of this study was to asses the kinetics of cytokine release from COPD parenchymal explants using an ex vivo model of lipopolysaccharide (LPS) induced acute inflammation. METHODS: Lung tissue from 24 patients classified by the GOLD guidelines (7F/17M, age 67.9 +/- 2.0 yrs, FEV1 76.3 +/- 3.5% of predicted) and 13 subjects with normal lung function (8F,5M, age 55.6 +/- 4.1 yrs, FEV1 98.8 +/- 4.1% of predicted) was stimulated with 100 ng/ml LPS alone or in combination with either neutralising TNFalpha or IL-10 antibodies and supernatant collected at 1,2,4,6,24, and 48 hr time points and analysed for IL-1beta, IL-5, IL-6, CXCL8, IL-10 and TNFalpha using ELISA. Following culture, explants were embedded in glycol methacrylate and immunohistochemical staining was conducted to determine the cellular source of TNFalpha, and numbers of macrophages, neutrophils and mast cells. RESULTS: In our study TNFalpha was the initial and predictive cytokine released followed by IL-6, CXCL8 and IL-10 in the cytokine cascade following LPS exposure. The cytokine cascade was inhibited by the neutralisation of the TNFalpha released in response to LPS and augmented by the neutralisation of the anti-inflammatory cytokine IL-10. Immunohistochemical analysis indicated that TNFalpha was predominantly expressed in macrophages and mast cells. When patients were stratified by GOLD status, GOLD I (n = 11) and II (n = 13) individuals had an exaggerated TNFalpha responses but lacked a robust IL-10 response compared to patients with normal lung function
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