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Gustavo S Figueiredo, Majlinda Lako, Simi Ali, Francisco C Figueiredo; Molecular analysis of inflammation in pannus tissue from patients with total limbal stem cell deficiency by quantitative real time PCR. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3468.
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© ARVO (1962-2015); The Authors (2016-present)
Total limbal stem cell deficiency (TLSCD) leads to complete conjunctivalization of the corneal surface with a pannus tissue. We aim to determine the levels of different markers of inflammation in this tissue by quantitative real time PCR.
The pannus tissue is excised from all patients undergoing autologous limbal stem cell transplantation (ALSCT) in our current clinical study. This tissue has been prospectively collected from consecutive patients undergoing this treatment (n=19). A literature review identified 42 genetic markers of ocular surface inflammation, which were used to produce a custom-designed 96-well qPCR plate (Qiagen Ltd). RNA was extracted from all samples (ReliaPrep™, Promega) and cleaned-up (RNA Clean & Concentrator™, Zymo Research), cDNA synthesised (Qiagen Ltd) and samples processed by qPCR. Four samples each from normal conjunctiva and pterygia were analysed as negative and positive controls. Results from TLSCD samples were statistically analysed in comparison with negative and positive controls. Correlations with clinical grades of inflammation (pain score, bulbar hyperemia) were investigated.
Thirteen pannus samples were analysed from 13 patients. Compared with normal conjunctiva, inflammatory markers were elevated in the TLSCD samples: TNF (p=0.0005), IL1B (p=0.003), IL8 (p=0.001), IL6 (p=0.05), IL10 (p=0.02) and CCL2 (p=0.02). Markers of epithelial cell proliferation and adhesion were reduced in TLSCD: FGF7 (p=0.003), OCLN (p=0.002) and ITGB1 (p=0.0003); as were TGFB2 (involved in cytokine inhibition, p=0.03) and FAS (a regulator of apoptosis, p=0.01). There was a positive correlation with pain for ITGA6 (r=0.64,p=0.02) and with hyperemia for IL12A (r=0.56,p=0.05).
In line with clinical evidence, the conjunctivalized corneal surface in TLSCD is highly inflamed and this is reflected by the elevation of common markers of inflammation in the pannus tissue and their positive correlation with pain and hyperaemia. Further work is needed to investigate the impact of these inflammation markers on the survival, proliferation and differentiation of LSCs using our explant culture system. Better understanding of the inflammation in this tissue may be of diagnostic value (in non-invasive analysis of markers in tears) and allow for preoperative improvement of the ocular surface to maximise LSC engraftment after ALSCT.
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