Investigation of â defensin gene expression in the ocular anterior segment by semiquantitative RT-PCR

Aim—To determine if â defensins are expressed in the anterior segment of the eye and to determine the temporal pattern of expression using a real time semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Methods—Ocular tissue (corneal epithelium, conjunctiva, iris, and lens capsule) was collected from 23 patients undergoing surgery. Serial corneal or conjunctival impression cytology was performed on a separate group of 10 patients undergoing corneal tunnel phacoemulsification or trabeculectomy. The samples were analysed for â defensin mRNA by semiquantitative RT-PCR and the mRNA standardised for cell numbers. Results—RT-PCR amplified â defensin 1 mRNA from all lens capsule (six) and corneal (five) samples and all but one of the conjunctival (six) and iris samples (six). â Defensin 2 mRNA was amplified from three of five corneal, two of six conjunctival, and none of the iris or capsule samples. The impression cytology samples demonstrated a decline in defensin expression over the three time points studied. There were no false positive results from either the no-RT or negative control samples. Conclusions—This preliminary study confirms that natural antibacterial peptides are expressed in the anterior segment of the eye. There appears to be a pattern to the expression with inducible â defensin 2 not expressed intraocularly and higher levels of â defensin 1 than â defensin 2 expressed in extraocular tissue. The implication is that â defensin 1 is constitutively produced in ocular tissues and represents a key component of the innate immune system. (Br J Ophthalmol 2000;84:523–526) Defensins are a family of highly conserved cysteine rich cationic proteins, which have an important role in combating microbial infection. They are found throughout nature, in plants as well as animals and have broad spectrum activity against Gram positive and Gram negative bacteria, as well as enveloped viruses and fungi. Defensins are attracted by the microbial membrane’s negative charge and cause disruption by first binding, then intercalating, to form pores that destroy the integrity of the lipid bilayer. This dissipates the membrane potential and kills the cell. Defensins have a synergistic action with known antibacterial components of aqueous humour and the precorneal tear film, such as lysozyme and lactoferrin, as well as with antibiotics. As defensins rely on the physical properties of the bacterial cell envelope, they may have an increasingly important role as antibiotic resistance becomes more prevalent. Two groups of defensins are recognised in humans: á defensins, produced by neutrophils and the Paneth cells of the small intestine, and â defensins secreted by epithelial, or epithelial derived tissues. The â defensin production at mucosal surfaces is an important part of the innate immune system. Two human â defensins have been described, human â defensin 1 (hBD-1) and human â defensin