Alison M. McDermott

A Review of Antimicrobial Peptides and Their Therapeutic Potential as Anti-Infective Drugs

Purpose: Antimicrobial peptides (AMPs) are an essential part of innate immunity that evolved in most living organisms over 2.6 billion years to combat microbial challenge. These small cationic peptides are multifunctional as effectors of innate immunity on skin and mucosal surfaces and have demonstrated direct antimicrobial activity against various bacteria, viruses, fungi, and parasites. This review summarizes their progress to date as commercial antimicrobial drugs for topical and systemic indications. Methods: Literature review. Results: Despite numerous clinical trials, no modified AMP has obtained Food & Drug Administration approval yet for any topical or systemic medical indications. Conclusions: While AMPs are recognized as essential components of natural host innate immunity against microbial challenge, their usefulness as a new class of antimicrobial drugs still remains to be proven.

Toll-like receptors in ocular surface disease.

The ability of the ocular surface to mount an immune response is in part attributed to a family of proteins called toll-like receptors (TLRs). The latter are evolutionary conserved receptors that recognize and respond to various microbes and endogenous ligands. In addition to their recognition function, TLR activation triggers a complex signal transduction cascade that induces the production of inflammatory cytokines and co-stimulatory molecules, thus initiating innate and adaptive immunity. Toll-like receptor expression at the ocular surface is modulated during infection (e.g. Herpes simplex, bacterial keratitis and fungal keratitis) as well as during various inflammatory conditions (allergic conjunctivitis and dry-eye syndrome). Here recent findings regarding TLR expression and their involvement in various ocular surface diseases are discussed.

Human ß-defensin 2 is up-regulated during re-epithelialization of the cornea

Purpose. Human ß-defensins 1 and 2 (hBD-1, -2) are antimicrobial peptides found in several epithelia including corneal epithelium. Breach of the epithelium leaves the cornea vulnerable to infection so we sought to determine if there is an increase in defensin expression after injury. Methods. The epithelium from human cadaver corneas was collected by scraping (original samples). The corneas were then placed into organ culture to permit regeneration of the epithelium. Samples of re-grown epithelium were collected when healing was partially and 100% complete as determined by fluorescein staining. Total RNA was extracted from original and re-grown samples and used in RT-PCR reactions using primers specific for hBD-1 and hBD-2 and the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase. Immunoblotting was performed to detect defensin peptide in original and re-grown samples. Results. hBD-1 mRNA was detected in all original epithelial tissue samples (n = 10) examined suggesting that it is constitutively expressed. hBD-2 mRNA was detectable in only two of the ten samples. Of six corneas placed in to organ culture, hBD-1 mRNA expression was unchanged in the re-grown epithelial samples compared to the original epithelium samples, however the expression of hBD-2 mRNA was markedly increased. hBD-1 and hBD-2 peptides showed the same pattern of expression as their respective transcripts. Conclusions. These data show that hBD-2 mRNA and peptide is up-regulated in the corneal epithelium during re-epithelialization which is in keeping with the role of this defensin as an antimicrobial peptide.

Toll-like receptor activation modulates antimicrobial peptide expression by ocular surface cells.

The ability of the ocular surface to respond to pathogens is in part attributed to toll-like receptors (TLRs) that recognize conserved motifs on various microbes. This study examines TLR expression on various ocular surface cells, if TLR agonists can modulate the expression of antimicrobial peptides (AMPs), human beta defensins (hBD) and cathelicidin (hCAP-18/LL-37) which maybe functionally active against Pseudomonas aeruginosa (PA) and if TLR agonists or AMPs can modulate TLR mRNA expression. TLR1-10 mRNA expression was examined in corneal epithelial, corneal stromal cells and conjunctival epithelial cells by RT-PCR. To confirm protein expression flow cytometry or immunostaining was performed for selected TLRs on some cell cultures. Ocular surface cells were cultured with a range of TLR agonists and then hBD-1, 2, 3, or hCAP-18 mRNA and protein expression was determined by RT-PCR and immunoblotting. In some experiments, cells were cultured with a cocktail of agonists for TLR3, 5 and 6/2 and the antimicrobial activity of the culture media was tested against PA. TLR mRNA expression was also examined in primary human corneal epithelial cells (HCEC) treated with either 3 μg/ml of hBD-2, 5 μg/ml of LL-37 or TLR4, 5 and 9 agonists. Overall, the ocular surface cells expressed mRNA for most of the TLRs but some differences were found. TLR2 was not detected in corneal fibroblasts, TLR4 was not detected in primary cultured or freshly isolated HCEC, TLR5 was not detected in conjunctival epithelial cells (IOBA-NHC) and corneal fibroblasts, TLR7 was not detected in freshly isolated HCEC and TLR10 was not detected in HCEC and IOBA-NHC. TLR8 mRNA was not expressed by any of the samples tested. Immunostaining of cadaver corneas revealed TLR5 and 9 expression throughout the cornea while TLR3 was significantly expressed only in the epithelium. Flow cytometry and immunostaining revealed cultured fibroblasts expressed TLR9 but had no significant TLR3 expression. hBD-2 expression was upregulated by TLR1/2, 3, 4, 5 and 6/2 agonists depending on the cell type, whereas only the TLR3 agonist upregulated the expression of hCAP-18 in primary HCEC. The combination of TLR3, 5 and 6/2 agonists in primary HCEC, upregulated hBD-2 and hCAP-18 mRNA and peptide expression and secretion into the culture media, which significantly killed PA. This antimicrobial activity was primarily attributed to LL-37. TLR agonists did not modulate TLR expression itself, however, LL-37 or hBD-2 downregulated TLR5, 7 and/or 9 mRNA depending on the cell type. TLRs are expressed on the ocular surface and TLR agonists trigger the production of LL-37 and hBD-2, with LL-37 being particularly important for protecting the ocular surface against PA infection.

Antimicrobial peptides and wound healing: biological and therapeutic considerations

Repair of tissue wounds is a fundamental process to re‐establish tissue integrity and regular function. Importantly, infection is a major factor that hinders wound healing. Multicellular organisms have evolved an arsenal of host‐defense molecules, including antimicrobial peptides (AMPs), aimed at controlling microbial proliferation and at modulating the hosts immune response to a variety of biological or physical insults. In this brief review, we provide the evidence for a role of AMPs as endogenous mediators of wound healing and their promising therapeutic potential for the treatment of non‐life‐threatening skin and other epithelial injuries.

Dimensions and Morphology of the Cornea in Three Strains of Mice

PURPOSE To use a histologic approach to obtain dimensional and morphologic information on the cornea in three commonly used strains of mice. METHODS Adult mice (three each of 129/SVJ, C57BL/6, and BALB/c) were euthanatized, and the eyes were enucleated, immersed in 2% glutaraldehyde fixative, and prepared for light and transmission electron microscopy. The full corneal, epithelial, stromal, and posterior limiting lamina (PLL) with endothelium thicknesses were measured at the same location centrally and peripherally. RESULTS All three strains showed a statistically significant (P < 0.001) decrease in overall thickness in the peripheral compared with the central cornea. The decrease was due to a reduced thickness of both the epithelium and the stroma. The stroma and epithelium contributed to approximately two thirds and one third of the total corneal thickness, respectively. The epithelium had the classic stratified layout and consisted of 13.00 +/- 1.41 layers centrally versus 10.33 +/- 1.37 peripherally. Some adaptation of stromal tissue was found immediately adjacent to the epithelial basement membrane, but a clearly defined anterior limiting lamina did not exist. The stroma was organized into lamellae but lacked the anterior branching and interweaving reported in humans and had unmyelinated nerve fibers within micrometers of the endothelium. The PLL was 2.17 +/- 0.3 microm thick and was divided into pre- and postnatal layers, with striated bodies in the postnatal portion. CONCLUSIONS This study demonstrated that in the three strains of mice examined, the cornea becomes significantly thinner toward the periphery. Dimensionally, proportionally, and anatomically the three strains used appeared to be similar. However, morphologic differences were observed compared with other mammals, and awareness of these differences is important when using the mouse as an animal model applicable to the human.

The TFOS International Workshop on Contact Lens Discomfort: Report of the contact lens interactions with the ocular surface and adnexa subcommittee

Efron, N., Jones, L., Bron, A. J., Knop, E., Arita, R., Barabino, S., … Markoulli, M. (2013). The TFOS International Workshop on Contact Lens Discomfort: Report of the Contact Lens Interactions With the Ocular Surface and Adnexa Subcommittee. Investigative Opthalmology & Visual Science, 54(11), TFOS98. https://doi.org/10.1167/iovs.13-13187

Pathways of corneal and ocular surface inflammation: A perspective from the Cullen symposium

The goal of this symposium was to coalesce information presented by 22 investigators in the field of corneal and ocular surface inflammation into common pathways of inflammation. The perspective elucidated in this article defines the components of the normal ocular surface immune architecture and describes the consensus reached on the mechanisms/pathways involved in 1) acute inflammation; 2) late-stage (chronic) response; and 3) allergic disease. Seven diagrams didactically illustrate mechanisms. This paper is the introductory article in a supplement containing 18 articles by the symposium participants.

In vitro activity of human β-defensin 2 against Pseudomonas aeruginosa in the presence of tear fluid

ABSTRACT Pseudomonas aeruginosa causes vision-threatening keratitis and is difficult to treat due to emerging resistance. Human β-defensin 2 (hBD-2) is an antimicrobial peptide expressed by ocular surface epithelia with broad-spectrum activity against various pathogens, including P. aeruginosa. The activity of hBD-2 against P. aeruginosa in the presence of human tears or NaCl was studied. In some experiments, tears were heat-inactivated, filtered, and separated into cationic/anionic fractions or mucin MUC5AC was removed by immunoprecipitation before use. Immunoprecipitation was performed to study the interaction between hBD-2 and MUC5AC. hBD-2 activity was reduced by 40 to 90% in the presence of 17.5 to 70% (vol/vol) tears. NaCl reduced hBD-2 activity, but at most it could account for only 36% of the inhibitory effect of tears. Heat inactivation and filtration attenuated the ability of tears to inhibit hBD-2 activity by 65 and 68%, respectively. Anionic tear fractions significantly reduced (86%) the activity of hBD-2, whereas only a 22% reduction was observed with the cationic fractions. In the absence of MUC5AC, the activity of hBD-2 was restored by 64%. Immunoprecipitation studies suggested that the loss of hBD-2 activity in tears is due to a direct binding interaction with MUC5AC. Our data showed that the antimicrobial activity of hBD-2 is sensitive to the presence of human tears and that this is partly due to the salt content and also the presence of MUC5AC. These data cast doubt on the effectiveness of hBD-2 as an antimicrobial peptide, and additional studies are required to conclusively elucidate its role in innate immunity at the ocular surface in vivo.

The diagnosis and characteristics of moderate dry eye in non-contact lens wearers

Purpose. To identify and characterize moderate dry eye in non–contact lens wearers with a new scoring system-based dry eye questionnaire and to determine which objective tests better differentiate patients with moderate dry eye from healthy patients. Methods. Fifty-two healthy subjects (21 women and 31 men with a mean age of 27.8 ± 9.2 years) and 37 subjects with moderate dry eye (33 women and 4 men with a mean age of 36.4 ± 12.9 years) completed a 42-item dry eye questionnaire. Seventeen healthy subjects (11 women and 6 men with a mean age of 30.5 ± 9.7 years) and 28 subjects with moderate dry eye (24 women and 4 men with a mean age of 38.50 ± 3.8 years) underwent additional objective assessment of ocular surface health, tear osmolality, tear stability, and tear volume. Results. Subjects with moderate dry eye scored significantly higher (49.8 ± 20.3, P<0.0001) on the dry eye questionnaire than did normal subjects (11.7 ± 10.3). Ocular irritation symptoms worsened with progression of time of day in both groups of subjects. Internal reliability (0.95 Cronbach α) was excellent, and concurrent validity (Spearman ρ 0.507) was acceptable when compared to the McMonnies and Ho dry eye questionnaire. Significant differences in tear osmolality (P<0.00001), invasive tear breakup time (P<0.034), and corneal vital dye staining (P<0.0001) were detected between the two groups of subjects. A stepwise linear regression on objective clinical tests, however, did not account for 77% of the total variance in the questionnaire scores. Conclusions. A unique scoring system-based dry eye questionnaire was validated to separate non–contact lens wearers with moderate dry eye from healthy subjects. Objective tests of tear osmolality and stability and ocular surface integrity were better than other clinical measures at identifying differences between the two subject groups. The results strongly support the evidence that the diagnosis and treatment of moderate dry eye requires a detailed assessment of self-perceived symptoms and that objective clinical testing alone may be insufficient.