HLA-DR expression as a biomarker of inflammation for multicenter clinical trials of ocular surface disease

Abstract

There are currently no validated minimally invasive objective metrics for the classification and evaluation of ocular surface diseases and/or for evaluating treatment efficacy. We thus sought to establish a standardized methodology for determining the relative amount of the inflammatory biomarker HLA-DR on the ocular surface and to evaluate the precision, reliability and repeatability of its use for large multicenter clinical trials and translational research studies of ocular surface disease. Multiple studies were conducted to establish a Standard Operating Procedure (SOP) for utilizing HLA-DR expression as a minimally invasive, objective, ocular surface inflammatory biomarker. The established SOPs provide specific guidelines for HLA-DR collection and analysis, in order to incorporate it reliably into multicenter clinical trials and/or translational research. Duplicate cell samples from impression cytology (IC) samples of both normal and dry eye individuals were collected and split to assess repeatability (between the splits and between the duplicate samples). To determine storage capability, one duplicate was stained immediately and the other after 30 days cold storage. To demonstrate the feasibility of the use of the SOP for a multicenter clinical trial, clinicians out-of-state were trained to collect IC samples, and the samples shipped to our Biomarker Laboratory, logged, processed and analyzed. Demonstration of the ability to incorporate of IC into a randomized double masked clinical trial of dry eye disease (DED) was performed. In all cases, processing and analyses were performed by a masked independent observer. The validity/viability of the SOPs was established by demonstrating that: 1) sufficient numbers of cells can be collected via IC; 2) the precision/ repeatability of the relative biomarker expression quantified in samples; 3) personnel at distant sites can be taught to collect, store and ship samples successfully; 4) samples can be stored for up to 30 days (refrigeration) before processing without affecting results; 5) IC can be incorporated Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Presented in part at the 2010 annual meeting of the Association for Research in Vision and Ophthalmology. into a double blind randomized clinical trial (RCT) of DED; and 6) the Biomarker Laboratory can track a large number of masked samples reliably. In conclusion, our standard operating procedure for impression cytology analysis of HLA-DR expression appears to be repeatable and reproducible for use in multicenter clinical trials, providing a minimally invasive objec...

Effects of Dry Eye Therapies on Environmentally Induced Ocular Surface Disease

“…23,24 This marker was chosen because it was found to be elevated in the conjunctiva of dry eye patients and a significant decrease was observed in subjects treated with either topical cyclosporin A or oral polyunsaturated fatty acids. 25-26 Impression cytology was performed to obtain cells from the nasal bulbar conjunctiva at the enrollment (visit 1) and after each of the 90-minute low humidity challenges (visits 2-4). Levels of HLA-DR RNA transcripts significantly increased after the initial low humidity challenge at visit 2 (P = 0.04).…”

The Pathophysiology of Dry Eye Disease

“…118, 119 Disrupted immune tolerance in dry eye 112–114 elicits leads to dendritic cell maturation 120 and generation of autoreactive T effector cells 70, 101, 121–124 in mouse dry eye models. Human dry eye patients have an increased number of conjunctival dendritic cells 125, 126 and a higher percentage of cells expressing the dendritic cell maturation marker HLA-DR. 127–130 Depletion of dendritic cells prevented the development of dry eye disease in mice subjected to desiccating stress. 120 Mature dendritic cells prime antigen-specific Th1 and Th17 effector T cells in the conjunctival draining lymph nodes.…”

Image-guided evaluation and monitoring of treatment response in patients with dry eye disease

“…[39-41] Thus there is an urgent need for the development of new biomarkers for DED that are reliable, reproducible, consistent with symptoms, and reflect the underlying pathogenesis and severity of disease. [42] Herein we discuss a novel, non-invasive in vivo imaging approach that can potentially emerge as a tool for quantitative evaluation of disease and monitoring of treatment efficacy in DED, supplementing the current standard diagnostic tests. Through quantitative impressions of in vivo confocal micrographs based on densities of the subepithelial corneal immune cells, palpebral conjunctival immune cells and sub-basal nerves, when taken together with clinical diagnostic tests, in vivo imaging may enhance the clinical management of dry eye disease.…”