An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation

Truls Gråberg,1 Lovisa Strömmer,1 Erik Hedman,2 Mehmet Uzunel,3 Ewa Ehrenborg,4 Ann-Charlotte Wikström5 1Division of Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, 2Department of Clinical Pharmacology, Karolinska Uni...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Gråberg T, Strömmer L, Hedman E, Uzunel M, Ehrenborg E, Wikström AC
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2015
Materias:	Pathology RB1-214 Therapeutics. Pharmacology RM1-950
Acceso en línea:	https://doaj.org/article/486f91c8c53e468ca9b583f8a58f109d
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:486f91c8c53e468ca9b583f8a58f109d
record_format	dspace
spelling	oai:doaj.org-article:486f91c8c53e468ca9b583f8a58f109d2021-12-02T08:32:06ZAn ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation1178-7031https://doaj.org/article/486f91c8c53e468ca9b583f8a58f109d2015-08-01T00:00:00Zhttp://www.dovepress.com/an-ex-vivo-rt-qpcr-based-assay-for-human-peripheral-leukocyte-responsi-peer-reviewed-article-JIRhttps://doaj.org/toc/1178-7031Truls Gråberg,1 Lovisa Strömmer,1 Erik Hedman,2 Mehmet Uzunel,3 Ewa Ehrenborg,4 Ann-Charlotte Wikström5 1Division of Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, 2Department of Clinical Pharmacology, Karolinska University Hospital, 3Division of Therapeutic Immunology, Department of Laboratory Medicine, 4Atherosclerosis Research Unit, Department of Medicine, Solna, 5Unit of Translational Immunology, Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden Introduction: An assay to determine glucocorticoid (GC) responsiveness in humans could be used to monitor GC non-responsiveness in states of GC insufficiency and could provide a tool to adapt GC treatment to individual patients. We propose an ex vivo assay to test GC responsiveness in peripheral leukocytes. The assay was evaluated in a human experimental model of surgery-induced inflammation. Patients and methods: Changes in expression of the GC-regulated genes GILZ, IL1R2, FKBP5, and HLA-DR and glucocorticoid receptor alpha (GRα) were determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral leukocytes from surgical patients and healthy blood donors (total n=60) in response to low (1 nM) and high (1 µM) dexamethasone (DEX). The final selection of a suitable endogenous control gene was based on the studies of stability during DEX treatment and inflammation. Correlations between pre- and postoperative GC-induced gene expression, the postoperative systemic inflammatory and metabolic response (CRP, IL-6, white blood cell count, cytokines, resistin, free fatty acids, glucose, insulin, and adiponectin), and the clinical outcome were analyzed. The length of stay in the intensive care unit (ICU-LOS), the length of stay in the hospital, and postoperative complications were used to measure clinical outcome. Results: When the blood donors were compared to the patients, there were no significant differences in the regulation of the genes in response to DEX, except for GRα. Preoperative, but not postoperative, gene regulation of GILZ and GRα was negatively correlated to ICU-LOS (P<0.05 and P<0.01, respectively). Preoperative GILZ and FKBP5 gene regulation was negatively correlated to postoperative systemic TNFα and MIP-1α levels. Conclusion: We suggest that this assay could be used to determine GC responsiveness. An alteration in preoperative GC responsiveness may be related to a patient's ability to recover from surgically induced inflammatory stress. Keywords: glucocorticoid responsiveness, gene regulation, clinical outcome, GILZ, GRα, cytokinesGråberg TStrömmer LHedman EUzunel MEhrenborg EWikström ACDove Medical PressarticlePathologyRB1-214Therapeutics. PharmacologyRM1-950ENJournal of Inflammation Research, Vol 2015, Iss default, Pp 149-160 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Pathology RB1-214 Therapeutics. Pharmacology RM1-950
spellingShingle	Pathology RB1-214 Therapeutics. Pharmacology RM1-950 Gråberg T Strömmer L Hedman E Uzunel M Ehrenborg E Wikström AC An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation
description	Truls Gråberg,1 Lovisa Strömmer,1 Erik Hedman,2 Mehmet Uzunel,3 Ewa Ehrenborg,4 Ann-Charlotte Wikström5 1Division of Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, 2Department of Clinical Pharmacology, Karolinska University Hospital, 3Division of Therapeutic Immunology, Department of Laboratory Medicine, 4Atherosclerosis Research Unit, Department of Medicine, Solna, 5Unit of Translational Immunology, Department of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden Introduction: An assay to determine glucocorticoid (GC) responsiveness in humans could be used to monitor GC non-responsiveness in states of GC insufficiency and could provide a tool to adapt GC treatment to individual patients. We propose an ex vivo assay to test GC responsiveness in peripheral leukocytes. The assay was evaluated in a human experimental model of surgery-induced inflammation. Patients and methods: Changes in expression of the GC-regulated genes GILZ, IL1R2, FKBP5, and HLA-DR and glucocorticoid receptor alpha (GRα) were determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral leukocytes from surgical patients and healthy blood donors (total n=60) in response to low (1 nM) and high (1 µM) dexamethasone (DEX). The final selection of a suitable endogenous control gene was based on the studies of stability during DEX treatment and inflammation. Correlations between pre- and postoperative GC-induced gene expression, the postoperative systemic inflammatory and metabolic response (CRP, IL-6, white blood cell count, cytokines, resistin, free fatty acids, glucose, insulin, and adiponectin), and the clinical outcome were analyzed. The length of stay in the intensive care unit (ICU-LOS), the length of stay in the hospital, and postoperative complications were used to measure clinical outcome. Results: When the blood donors were compared to the patients, there were no significant differences in the regulation of the genes in response to DEX, except for GRα. Preoperative, but not postoperative, gene regulation of GILZ and GRα was negatively correlated to ICU-LOS (P<0.05 and P<0.01, respectively). Preoperative GILZ and FKBP5 gene regulation was negatively correlated to postoperative systemic TNFα and MIP-1α levels. Conclusion: We suggest that this assay could be used to determine GC responsiveness. An alteration in preoperative GC responsiveness may be related to a patient's ability to recover from surgically induced inflammatory stress. Keywords: glucocorticoid responsiveness, gene regulation, clinical outcome, GILZ, GRα, cytokines
format	article
author	Gråberg T Strömmer L Hedman E Uzunel M Ehrenborg E Wikström AC
author_facet	Gråberg T Strömmer L Hedman E Uzunel M Ehrenborg E Wikström AC
author_sort	Gråberg T
title	An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation
title_short	An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation
title_full	An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation
title_fullStr	An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation
title_full_unstemmed	An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation
title_sort	ex vivo rt-qpcr-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation
publisher	Dove Medical Press
publishDate	2015
url	https://doaj.org/article/486f91c8c53e468ca9b583f8a58f109d
work_keys_str_mv	AT grabergt anexvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT strommerl anexvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT hedmane anexvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT uzunelm anexvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT ehrenborge anexvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT wikstromac anexvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT grabergt exvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT strommerl exvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT hedmane exvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT uzunelm exvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT ehrenborge exvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation AT wikstromac exvivortqpcrbasedassayforhumanperipheralleukocyteresponsivenesstoglucocorticoidsinsurgicallyinducedinflammation
_version_	1718398480581394432

An ex vivo RT-qPCR-based assay for human peripheral leukocyte responsiveness to glucocorticoids in surgically induced inflammation

Ejemplares similares