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B. Stampachiacchiere, A. Micera, M. Sacchetti, A. Iovieno, C. Cantera, A. Lambiase, S. Bonini; Impression Cytology as a Tool for Investigating Toll–Like Receptor Expression in the Ocular Surface . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2269.
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© ARVO (1962-2015); The Authors (2016-present)
the toll like receptor (TLR) are involved in immune function and are over–expressed by human corneal and conjunctival epithelium during microbial infections or allergic inflammation. To propose a simple method to evaluate TLR expression in conjunctival impression cytologies of healthy subjects and patients with VKC.
Conjunctival impression cytologies from VKC (n=9) and healthy (n=9) subjects were processed according to three different RNA extraction methods (GENTRA, MIRVANA, QIAGEN). Relative Real–Time PCR for TLR–4 and TLR–9 was carried out according to a standard procedure. TLR–4 and TLR–9 PCR product specificity was confirmed by melting curve examination during amplification. ANOVA analysis was performed to compare either the total RNA recovery by the different technique or TLR–4 and TLR–9 expression between VKC and controls.
3 ug total RNA were obtained from all samples without any significant difference among extraction techniques. TLR4 and TLR9mRNA were detected in all healthy impression cytologies. TLR9mRNA was significantly down–regulated in VKC cytologies as compared to healthy ones. Up–regulation of TLR4mRNA was observed only in three out of five evaluated patients with VKC, that were not taking anti–allergic medicals.
we propose the impression cytology processed by real–time PCR to investigate ocular surface features and suggest that TLRs expression may represent an additional tool to diagnosis/monitoring VKC. TLR9 data is in line with our previous study showing down–regulation of TLR–9 expression in VKC conjunctival biopsy, supporting the hypothesis of the strength TLR9 involvement in VKC pathogenesis. The variability in TLR4 expression suggests that this TLR may be influenced by anti–allergic treatment and/or by the severity of the disease. This simple and quantitative method might be extended as well to diagnosis/monitoring a wild spectrum of ocular surface diseases.
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