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Andrea Leonardi, Philippe Daull, Jean-Sebastien Garrigue, Elena Tarricone, Mylene Docquier, Paola Brun; Conjunctival transcriptome changes in vernal keratoconjunctivitis. Invest. Ophthalmol. Vis. Sci. 2016;57(12):302.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the extent of the human transcriptome that can be quantified from conjunctival impression cytology samples. The aims are to determine if sufficient RNA can be isolated from a patient’s conjunctiva, to identify differences in gene expression between vernal keratoconjuctivits patients (VKC) and normal subjects (CT), and to identify potential disease or disease activity biomarkers.
Conjunctival cells were collected by impression cytology (Eyeprim) from 23 VKC patients and 8 CT. Based on prior optimization of all assay steps, RNA was isolated from the samples using a Qiagen RNeasy Plus Mini Kit. The RNA integrity number (RIN) was assessed with an Agilent Bioanalyzer. Samples from CT and VKC patients were then assayed with the NanoString human immunology codeset to assess the expression levels of 579 immunology-related genes.
The inflammatory NanoString assay on 579 genes showed that more than 200 genes were significantly overexpressed in VKC compared to CT samples. Of these, more than 70 genes differed by a factor of 3 between the VKC and CT, and 20 by a factor of >20. Of the most overexpressed genes in the VKC group, to be noted were CCL13, CCL18, CCL24, CXCL1, CLEC4E, CLEC7A, CD28, CSF3R, FN, GATA3, ICAM1-2-4, IL-5, IL-9, IL-13, IL-23, ITGA2B, ITLN1, KLRC3, LILRB, MAF, MUC1, NOD2, PTAFR, TGFbeta1, TLR8 and ZAP70. 23 genes were significantly down-regulated in VKC, including CASP1 (CASP3 and CASP8 were increased 1.5 fold) CCL20, CFD, CR2, CX3CR1, IL-18 (IL-18Rs were all up-regulated), LTF, TLR5, HLA-DQB1 and VCAM1.
Conjunctival impression cytology can be used to collect sufficient RNA from conjunctival surface cells that, when processed optimally, allows successful transcriptome-wide expression analysis. Factors involved in both innate and adaptive arms of the immune system were found over-expressed in VKC samples. The increased expression of several chemotactic factors and co-stimulatory signals required for T cell activation and survival, confirms that VKC is mostly a cell-mediated pathology. While the present transcriptome analysis used a limited number of patients, larger studies with different types and severities of ocular allergy should reveal significant gene expression trends that can then be targeted to improve ocular allergy treatments.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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