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P. Versura, V. Profazio, C. Coslovi, M. Fresina, E. C. Campos; Hyperosmolar Stress is Related to HLA-DR Overexpression in Non Sjogren’s Dry Eye Patients and in Cultured Human Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1917.
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To determine whether there is a link between tear hyperosmolarity and the initiation of the immune response by the conjunctival epithelium. The expression and distribution of HLA-DR were evaluated in mild and moderate dry eye (DE) patients and in anin vitro model
15 normal subjects (15 eyes) and 25 DE patients (32 eyes), diagnosed and scored 1-3 according to DEWS severity system were included in the study. Tear osmolarity was recorded with TearLab Osmolarity System (OcuSense), conjunctival cytology samples were obtained by scraping, to evaluate inflammation, and imprint at the nasal bulbar area.Subconfluent primary human conjunctival epithelial cells (pHCECs) and human conjunctival organ cultures (hCOCs) were obtained from donor eyes (n=5), cultured in iso-osmolar medium (305 mOsm/L) and exposed for 24 hrs to media with progressively higher osmolarity. HLA-DR expression was estimated by immunocytochemistry on imprints from normal subjects and DE patients, on pHCECs, on formalin fixed-paraffin embedded hCOCs; results were expressed as % positive cells vs total or cells/mm2. Statistical evaluation was performed by applying the unpaired Student’s t-test, the Spearman’s rho and the Pearson’s r correlation coefficients (significance p<0,05).
HLA-DR expressed differently in dry eye vs control subjects (% mean + SD respectively 46,16 + 7,2 vs. 7,48 + 1,14, p< 0,0001) and exhibited significantly high correlations vs tear osmolarity value (r 0,614, p<0,0001), scraping cytology inflammatory score (r 0,692, p<0,0001), imprint score (rho 0,583, p<0,001).In vitro experiments showed a progressive increase in HLA-DR expression as the media’s osmolarity was increased, from 6,75 + 1,16 (% mean+SD) in cells cultured in normal osmolarity to 9,96 + 1,37 and 12,94 + 4,04 in cells cultured in 350 and 400 mOsM/L media, respectively (p<0,05). A progressive increase was also found in hCOCs: from 195 + 58,6 (cells/mm2+SD) in iso-osmolar medium to 277,8 + 37,9 and 300,8 + 67,3 in media with the range of osmolarities as above, respectively (p<0,05).
Expression of HLA-DR in hCECs appears to be correlated with elevated tear osmolarity in DE patients and the results are confirmed in in vitro models. The present study suggests extracellular hyperosmolarity may induce inflammation in conjunctival cells.
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