A Abedin

A Novel Antimicrobial Peptide on the Ocular Surface Shows Decreased Expression in Inflammation and Infection

PURPOSE Antimicrobial peptides (AMPs) are cationic host defense peptides with microbicidal and cell-signaling properties. They show promise as potential therapeutic agents. In the present study, a beta-defensin AMP gene was isolated from the ocular surface for the first time, and its expression was characterized in the presence of ocular inflammation and/or infection. METHODS Total RNA was obtained from impression cytology samples of the conjunctiva and cornea of normal patients and of those with bacterial, viral, acanthamoeba, or dry eye disease. The expression of the beta-defensin AMP DEFB-109 was determined by using reverse transcription-polymerase chain reaction (RT-PCR). Relative quantification of the gene in the various groups was performed by means of real-time PCR. RESULTS DEFB-109 was constitutively expressed in all samples. The gene showed significantly decreased expression in the presence of all types of inflammation/infection. Reduced expression featured most prominently in acanthamoeba infection; the least change from normal was in dry eye. CONCLUSIONS The discovery of DEFB-109 on the ocular surface enhances our knowledge of the profile of AMPs at this important mucosal surface. The fact that its expression is significantly reduced in both inflammatory and infective ocular surface disease reflects not only an intimate balance between this host defense gene and microbes but indicates a role other than purely microbicidal. This discovery will enable the mechanisms behind the intriguing phenomenon of reduced gene expression of an AMP in disease states to be uncovered.

Alcohol delamination of the corneal epithelium for recalcitrant recurrent corneal erosion syndrome: a prospective study of efficacy and safety

Aim: To investigate the safety and efficacy of alcohol delamination of the corneal epithelium for recalcitrant recurrent corneal erosion syndrome (RCE) in a prospective non-comparative interventional case series. Methods: Patients with RCE who remained symptomatic despite topical lubrication and 3-month use of an extended wear (bandage) contact lens were included. The intensity of pain on a scale of 1 to 10, and duration and frequency of attacks were recorded. Patients were treated with alcohol delamination and followed up at 1 week, 1 month and then at 12 months, which was the minimum follow up time. Success was defined as a resolution of symptoms after 1 month of treatment, and partial success as a significant reduction in symptoms manageable with topical lubricants, and failure where symptoms were unchanged or worsened. Results: 20 eyes of 20 patients with RCE caused by trauma (n = 14), anterior basement membrane dystrophy (n = 5) or idiopathic (1) were studied. The mean age of patients was 44 years and mean follow-up 24 months. Two eyes were lost to follow-up. 15 (83%) eyes were qualified as successes, 3 as partial successes and no failures were seen. No intraoperative complications were observed. 1 patient developed transient subepithelial haze that resolved with topical steroid, 1 had a change in refraction and 1 developed herpetic stromal keratitis 2 months postoperatively, which was successfully treated. No loss of vision was observed in any of the patients. Conclusion: Alcohol delamination of the corneal epithelium is a safe and effective method for treating patients with recalcitrant RCE.

Antimicrobial peptides expression by ocular surface cells in response to Acanthamoeba castellanii: an in vitro study

Aims Antimicrobial peptides (AMPs) are natural effectors of the innate immune response. Much work has been done to study their response and effects on bacterial and viral infection. Little if any information is available in relation to protozoal infections. The aim of the study was to comprehensively study the gene expression of the ocular AMPs in human corneal limbal epithelial cells stimulated with Acanthamoeba castellanii (AC). Methods Human corneal limbal epithelial cells were exposed to AC at different time points, up to 9 h, the genomic profile of the AMPs were analysed at these time point using real time PCR. corneal limbal epithelial cells not infected with AC were used as controls. Results Seven of the eight studied AMPs showed statistically significant upregulation in gene expression. Human beta Defensin 3 (hBD3) showed a very significant 10-fold upregulation in the exposed cells and Ribonuclease-7 (RNase-7) showed a very early and consistent increase. Human beta Defensin 1 (hBD1) was the only downregulated AMP. Conclusions The study data suggests a possible role of the AMPs in combating the amoebic infection at the ocular surface. Using AMPs singly or in combination is a promising avenue for further exploration in the treatment of the sight threatening Acanthamoeba keratitis.

Signalling pathways involved in ribonuclease-7 expression.

Antimicrobial peptides are host defence molecules that play a potential role in preventing infection at the epithelial surfaces. Ribonuclease (RNase)-7 has been shown to possess a broad spectrum of microbicidal activity against various pathogens. Here, we demonstrate that RNase-7 protein is localised to the superficial layers of ocular surface cells and increased in response to interleukin (IL)-1β, suggesting an active role during inflammation related to ocular surface infection. Signal transduction pathways involved in RNase-7 expression are unknown. Involvement of transforming growth factor β-activated kinase-1 (TAK-1) activated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway molecules [c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38] were studied because of their importance in infection and inflammation. Blocking the MAPKs resulted in inhibition of RNase-7 expression in response to IL-1β. However, RNase-7 induction by IL-1β was not affected by inhibiting the NF-κB signalling pathway. In conclusion, our results indicate that RNase-7 expression is specifically mediated via MAPKs but not NF-κB signalling pathways.

Increased expression of hepcidin and toll-like receptors 8 and 10 in viral keratitis.

Purpose: Antimicrobial peptides (AMPs) and toll-like receptors (TLRs) form part of the “chemical barrier” of the ocular surface to microbes. Evidence suggests that pathogen recognition by TLR releases AMPs, altering AMP-TLR profiles in pathological states. This study investigated ocular surface expression of AMP-TLRs in health and disease. Methods: Complementary DNA from conjunctival and corneal impression cytology samples was used for semiquantitative and quantitative polymerase chain reactions, to determine gene expression of 6 AMPs and TLRs-1-10, in healthy subjects and patients with bacterial (n = 6), viral (n = 6), Acanthamoeba (n = 3), or dry eye (n = 7) diseases. Results: Semiquantitative polymerase chain reaction showed variable AMP expression within groups and some expression patterns between groups, increased levels of LEAP (liver-expressed antimicrobial peptide)-1/hepcidin in viral disease, LEAP-2 in dry eye, and human beta defensin 3 in bacterial disease. There was no significant variability in TLR expression. Quantitative polymerase chain reaction showed significantly higher expression of LEAP-1 (P = 0.002) and TLR-8 (P = 0.023) and TLR-10 (P = 0.014) in viral keratitis and LEAP-2 (P = 0.034) in dry eye, versus controls. Conclusions: Increased expression of LEAP-1 and TLRs 8 and 10 in viral keratitis is novel; TLR-10 has not previously had a documented ligand. LEAP-2 may have a role in dry eye. Further studies will help to improve the understanding of these diseases and yield novel therapeutic interventions.

Choroidal detachment in perforated corneal ulcers: frequency and management

Aims: To determine the frequency of choroidal detachment (CD) in eyes with non-traumatic corneal ulcer perforation and, also, to assess the efficacy and safety of cyanoacrylate glue in sealing corneal perforations. Methods: 18 eyes of 17 patients were studied. Inclusion criterion was any patient with a non-traumatic perforated corneal ulcer. All patients had a thorough history taken and complete ophthalmic examination including B-scan ultrasonography. Patient demographics, presence of CD, and efficacy of corneal gluing were assessed. Results: Eight of the 18 eyes (44%) were documented to have a CD. Among perforations of >2 mm2, six eyes (75%) were documented to have CD compared with two eyes (20%) with perforations of ⩽2 mm2 (p = 0.054). No correlation could be determined between perforation duration and incidence of CD. Of the 15 eyes that underwent gluing, there were 13 successes (87%) and two failures (13%). Within the successes four patients (27%) required re-gluing because of infection (one patient) or progression of melt and glue loosening (three patients). Failure was the result of severe progression of melting (one patient) and a very large perforation (one patient). Conclusion: Choroidal detachment following corneal ulcer perforation is common and is more likely in larger corneal perforations. Preoperative B-scan should be considered in cases of large corneal perforations requiring therapeutic keratoplasty to document choroidal detachment, which if large may require drainage. Cyanoacrylate glue is an effective and safe method for sealing small corneal perforations. A vigil must be maintained for infection while the glue and bandage contact lens are in situ.