Abstract
Purpose: :
Squamous metaplasia occurs in severe ocular surface diseases, such as Stevens–Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP) and Sjögren’s syndrome (SS). It represents an important clinical problem in that it causes pathological keratinization of the ocular surface. The molecular mechanisms triggering squamous metaplasia in this setting are unknown. We have begun to examine the hypothesis that inflammatory mediators are key inducers of squamous metaplasia. Our studies have been facilitated by use of a squamous cell biomarker, SPRR1B (also called cornifin, SPRR1) which has been used previously to examine the induction of squamous metaplasia in response to other stimuli. To validate the use of SPRR1B as a biomarker for squamous metaplasia, we compared its expression in human patients with SS and normal age–matched controls.
Methods: :
SPRR1B gene expression was measured in 10 normal vs. 10 SS patients by quantitative, real–time PCR. RNA was extracted from conjunctival impression cytology specimens obtained using mixed cellulose ester membranes that were briefly applied to the temporal, bulbar conjunctiva and gently pulled away. Specimens were stored in RNAlater® until RNA extraction using the Qiagen RNeasy Micro Kit. The quality of RNA from all samples was assessed using the 2100 Agilent Bioanalyzer®. One hundred nanograms of total RNA was used for cDNA synthesis using the TaqMan reverse transcription kit from Applied Biosystems. Real–time PCR was carried out using a SYBR green system from Superarray Biosciences.
Results: :
SPRR1B expression is upregulated in human patients with SS. The average threshold cycles (Ct) values for GAPDH were used as an endogenous reference to correct for differences in amount of RNA added to each sample. Using real–time PCR comparative Ct values, our data showed a 3–fold increase in SPRR1B expression in SS patients (mean±SEM Ct = 0.3±0.1 vs. 0.9±0.3 in normals vs. SS, respectively).
Conclusions: :
We have identified a molecular biomarker for squamous metaplasia that is increased in human patients with ocular surface disease caused by SS. Validation of this biomarker in humans suggests that it represents a valuable marker that can be used to study the molecular mechanisms of squamous metaplasia in immune–mediated ocular surface disease. A better understanding of the molecular mechanisms underlying the induction of SPRR1B in patients with squamous metaplasia could ultimately lead to the identification of novel therapeutic targets to prevent pathological keratinization of the ocular surface.
Keywords: conjunctiva • autoimmune disease • gene/expression