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LC Huang, S Narayanan, RL Redfern, AM McDermott; Human Tears Inhibit the Antimicrobial Activity of Human beta-Defensin-2 In Vitro . Invest. Ophthalmol. Vis. Sci. 2002;43(13):82.
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Purpose: Human ß-defensin-2 (hBD-2) is an antimicrobial peptide whose expression by corneal and conjunctival epithelial cells is upregulated by infection and inflammatory cytokines. Studies have shown that hBD-2 antimicrobial activity is attenuated by increasing salt concentrations (50-200mM NaCl). Here we have investigated the effect of tears (approx. 150mM NaCl, 300 mOsm/kg) on hBD-2 antimicrobial activity against Pseudomonas aeruginosa (PA, ATCC #27853). Methods: Unstimulated tears were collected from subjects free of ocular surface disease using a glass capillary tube. Tear osmolality was measured using a Wescor Vapro-5520 vapour pressure osmometer. Mid log phase PA (10ul of 107cfu/ml) were incubated with hBD-2 in the absence or presence of different amounts of tears (final reaction volume 50ul) for 2 hrs at 37C. Nutrient broth agar plates were inoculated with 10ul of a 1 in 20 dilution of the reaction mixture and left to incubate for 24 hrs at 37C after which time the number of colonies was counted. Results: hBD-2 alone inhibited PA growth in a concentration dependent manner, with 100% inhibition being seen at 100ug/ml hBD-2. Tears alone were unable to inhibit the growth of PA. When tested in the presence of tears, the antimicrobial activity of 100ug/ml hBD-2 was markedly reduced. With a reaction mixture containing 17.5% v/v tears (osmolality 86±12 mOsm/kg) or 70% v/v tears (osmolality 206±11 mOsm/kg) the effectiveness of hBD-2 was reduced by 30% and 84% respectively (n=3). Conclusion: hBD-2 inhibits the growth of PA in vitrobut this activity is reduced in the presence of tears, thus agreeing with observations by others showing loss of hBD-2 antimicrobial activity with high salt concentrations. This brings in to question the effectiveness of hBD-2 at the ocular surface, although potential synergistic interactions with other antimicrobials insensitive to salt concentration need to be addressed.
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