Development and validation of a novel bioassay to determine glucocorticoid sensitivity.

Madeleine L. Stimpson,Ashwin D. Dhanda,Emily L. Williams,Richard W. Lee,Doyo G. Enki,Peter L. Collins,Ashish Sinha

doi:10.1186/s40364-016-0079-y

Abstract

BackgroundGlucocorticoids (GCs) remain the first line treatment for almost all non-infectious inflammatory diseases, ranging from acute asthma to rheumatoid arthritis. However, across all conditions, patients have a variable response to GCs with approximately 30% being non-responders. This group of GC resistant patients is typically exposed to high-dose GCs and their side-effects before more appropriate immunotherapy is instituted. Hence, there is a pressing clinical need for a predictive biomarker of GC responsiveness. The availability of such a tool would also enable patient stratification for the conduct of smart clinical trials in GC resistance. Lymphocyte GC sensitivity has been shown to be closely associated with clinical GC sensitivity in a number of inflammatory diseases. However, the method for determining in vitro GC response is not standardized and requires the use of specialist equipment, including a radioisotope to quantify cellular proliferation, making it challenging to translate into clinical practice.ResultsHere we describe the optimization and validation of a novel non-radioactive in vitro bioassay based on measuring cellular proliferation by incorporation of bromodeoxyuridine (BrdU), termed the BrdU incorporation in lymphocyte steroid sensitivity assay (BLISS). In comparison to the current gold standard lymphocyte GC sensitivity assay in 101 healthy control samples, BLISS has an area under receiver operating characteristic of 0.82 and a sensitivity of 83% for correctly identifying GC resistant subjects.ConclusionsThe performance of the novel BLISS bioassay makes it a strong candidate biomarker for clinical application. It now requires validation in a prospective patient cohort.Electronic supplementary materialThe online version of this article (doi:10.1186/s40364-016-0079-y) contains supplementary material, which is available to authorized users.

Highlights

Glucocorticoids (GCs) remain the first line treatment for almost all non-infectious inflammatory diseases, ranging from acute asthma to rheumatoid arthritis
Peripheral blood mononuclear cell (PBMC) were isolated by density gradient centrifugation over Ficoll-Paque Plus (VWR International, Lutterworth, Leicestershire, UK) using Leucosep tubes (Greiner BioOne International, Stonehouse, Gloucestershire, UK) and cell viability assessed by Trypan blue exclusion
There were no Dexamethasone Inhibition of Lymphocyte Proliferation Assay (DILPA) values excluded from the final analysis due to technical failure with low proliferation in PHA stimulated wells < 10,000 cpm

Summary

Introduction

Glucocorticoids (GCs) remain the first line treatment for almost all non-infectious inflammatory diseases, ranging from acute asthma to rheumatoid arthritis. GC resistant patients are both at risk of the consequences of their uncontrolled disease and, because they require higher and longer courses of therapy, they incur a greater burden of treatment side-effects. This could be avoided if alternative immunotherapeutics were to be instituted early in their disease course, but at present there is no way of identifying GC resistant individuals prior to GC treatment. They bind to cytoplasmic GC receptors, which translocate to the nucleus and bind to DNA at GC responsive elements

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