Inflammasomes, the eye and anti-inflammasome therapy.

Inflammasomes, key molecular regulators that play an important role in inflammation, consist of a central protein, an adaptor protein ASC (apoptosis speck-like protein) and a caspase-1 protein. Upon activation, caspase-1 induces maturation of cytokines such as interleukin-1ß (IL-1ß) and interleukin-18 (IL-18). The release of these cytokines can result in inflammation. Inflammasomes are activated by a variety of factors and their activation involves complex signalling leading to resolution of infection, but can also contribute to the pathology of inflammatory, autoimmune, and infectious diseases. The role of NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in the pathogenesis of ocular diseases such as glaucoma, age related macular degeneration (AMD), diabetic retinopathy, dry eye and infections of the eye has been established over the past decade. In experimental studies and models, inhibition of inflammasomes generally helps to reduce the inflammation associated with these eye diseases, but as yet the role of these inflammasomes in many human eye diseases is unknown. Therefore, a need exists to study and understand various aspects of inflammasomes and their contribution to the pathology of human eye diseases. The goal of this review is to discuss the role of inflammasomes in the pathology of eye diseases, scope for anti-inflammasome therapy, and current research gaps in inflammasome-related eye disease.

Interaction of the antimicrobial peptide melimine with bacterial membranes.

Melimine is a novel cationic peptide possessing broad-spectrum antimicrobial activity that is retained when attached to a surface, suggesting that interactions with bacterial membranes may be of primary importance to its activity. The effects of alterations in the environment on the conformation of melimine were investigated using circular dichroism and fluorescence spectra in membrane-mimetic solvents. Furthermore, the interactions of melimine with bacterial membranes of Pseudomonas aeruginosa and Staphylococcus aureus were examined using scanning electron and fluorescence microscopy, and perturbation of membrane integrity was tested by measurement of melimine-mediated diSC(3)-5 dye release from bacterial cells. Melimine has a predominantly random coil conformation that adopts a helical fold when exposed to organic solvents. However, when it is solubilised in micelles of sodium dodecyl sulphate, which are bacterial membrane-mimetic, the alpha-helical content increases to ca. 35-40%. A major effect of melimine was on the integrity of the cytoplasmic membrane both for P. aeruginosa and S. aureus. However, for P. aeruginosa the rapid loss of cytoplasmic membrane integrity correlated directly with loss of cell viability, whilst for S. aureus maximal dye release was obtained at concentrations where there was no significant loss of viability. There have been few studies to date investigating differences in the action of cationic peptides towards Gram-positive and Gram-negative bacteria. Consequently, further investigation of these mechanistic differences may allow more refined targeting of increasingly difficult-to-treat bacterial infections and/or further inform design of novel peptides with improved broad-spectrum activity.

Role of lactoferrin in the tear film.

The surface of the eye provides an inert barrier against infection. Through its unique combination of antimicrobial action and anti-inflammatory activities lactoferrin (Lf) in the tear film plays an important role in the maintenance of ocular health. In order to maintain clarity the eye must provide immunological defense without immunopathology. Along with physical barriers, soluble plasma factors and other proteins such as lysozyme, Lf produced by the acinar cells of the lacrimal gland serves a number of roles in defense for this purpose. Lf in tears provides antimicrobial efficacy by binding free iron thus reducing the availability of iron necessary for microbial growth and survival as well as pathogenesis. Lf has been shown to inhibit biofilm formation and thus may play a role in protecting contact lens surfaces from colonization. Virus particles' entry into epithelial cells is inhibited by Lf while an excess of Lf in tear film is thought to limit the opportunistic Lf-mediated bridging of adenovirus and host cell that occurs in other tissues. Lf dampens the classical complement activation pathway by binding to markers of inflammation and immune activation while pathogen-associated molecular patterns such as lipopolysaccharide (LPS) are targeted by Lf for removal through tears and hydrodynamic flushing. This review focuses on the role of Lf in human tear film and its contribution to ocular health during contact lens wear.

A novel cationic-peptide coating for the prevention of microbial colonization on contact lenses.

AIMS: To develop an antimicrobial peptide with broad spectrum activity against bacteria implicated in biomaterial infection of low toxicity to mammalian cells and retaining its antimicrobial activity when covalently bound to a biomaterial surface. METHODS AND RESULTS: A synthetic peptide (melimine) was produced by combining portions of the antimicrobial cationic peptides mellitin and protamine. In contrast to the parent peptide melittin which lysed sheep red blood cells at >10 microg ml(-1), melimine lysed sheep red blood cells only at concentrations >2500 microg ml(-1), well above bactericidal concentrations. Additionally, melimine was found to be stable to heat sterilization. Evaluation by electron microscopy showed that exposure of both Pseudomonas aeruginosa and Staphylococcus aureus to melimine at the minimal inhibitory concentration (MIC) produced changes in the structure of the bacterial membranes. Further, repeated passage of these bacteria in sub-MIC concentrations of melimine did not result in an increase in the MIC. Melimine was tested for its ability to reduce bacterial adhesion to contact lenses when adsorbed or covalently attached. Approximately 80% reduction in viable bacteria was seen against both P. aeruginosa and S. aureus for 500 microg per lens adsorbed melimine. Covalently linked melimine (18 +/- 4 microg per lens) showed >70% reduction of these bacteria to the lens. CONCLUSIONS: We have designed and tested a synthetic peptide melimine incorporating active regions of protamine and mellitin which may represent a good candidate for development as an antimicrobial coating for biomaterials. SIGNIFICANCE AND IMPACT OF THE STUDY: Infection associated with the use of biomaterials remains a major barrier to the long-term use of medical devices. The antimicrobial peptide melimine is an excellent candidate for development as an antimicrobial coating for such devices.

Role of quorum sensing by Pseudomonas aeruginosa in microbial keratitis and cystic fibrosis.

Pseudomonas aeruginosa is a ubiquitous bacterium that causes opportunistic infections in a range of host tissues and organs. Infections by P. aeruginosa are difficult to treat and hence there is interest in the development of effective therapeutics. One of the key mechanisms that P. aeruginosa uses to control the expression of many virulence factors is the N-acylated homoserine lactone (AHL) regulatory system. Hence, there is considerable interest in targeting this regulatory pathway to develop novel therapeutics for infection control. P. aeruginosa is the principal cause of microbial keratitis and of infections in cystic fibrosis (CF) sufferers, and AHL-dependent cell-to-cell signalling has been shown to be important for both infection types. However, keratitis tends to be an acute infection whereas infection of CF patients develops into a chronic, life-long infection. Thus, it is unclear whether AHL-regulated virulence plays the same role during these infections. This review presents a comparison of the role of AHL signalling in P. aeruginosa-mediated microbial keratitis and chronic lung infections of CF patients.

The effect of short term contact lens wear on the tear film and ocular surface characteristics of tolerant and intolerant wearers.

The purpose of this study was to determine the effect of contact lens wear on the tear film and ocular surface of people tolerant or intolerant to contact lens wear. Twenty subjects participated; 11 tolerants and nine intolerants. Their baseline tear film (no lens wear) was analysed with a range of clinical measurements and protein analyses (lactoferrin, sIgA and lysozyme). The tests were then repeated at the end of 6h of contact lens wear during the day and while lenses were worn. Both tolerants and intolerants showed statistically significant increases in bulbar and overall conjunctival redness after 6h of lens wear. For tolerants only, there was a statistically significant increase in the tear film meniscus area (0.08 mm(2) +/- 0.04 compared to 0.14 mm(2) +/- 0.06 (p = 0.023)) and a statistically significant decrease in the non-invasive tear film break-up time (NI-TBUT; 21.3 s +/- 5.7 compared to 3.7 s +/- 4.3 (p = 0.003)) after 6h of lens wear. There were no changes in other tear film or ocular surface parameters. The protein concentration and lipid layer appearance did not change during lens wear for either population. Prior to lens wear, tolerant subjects had a statistically longer NI-TBUT, higher phenol red thread test and higher tear flow rate. After 6h of lens wear and while wearing lenses, all but NI-TBUT remained statistically different. Lens wear affected only a small number of clinical variables and 6h wear did not effect the concentration of those proteins measured in tears in this study.

Non-steroidal anti inflammatory agents decrease bacterial colonisation of contact lenses and prevent adhesion to human corneal epithelial cells.

PURPOSE: To investigate non-steroidal anti-inflammatory agents (NSAIDs), salicylic acid, sodium diclofenac and ketorolac for inhibition of bacterial colonization of contact lenses (CL) and human corneal epithelial cells (HCE). METHODS: CLs pre-colonised with Pseudomonas aeruginosa, Haemophilus influenzae, Staphylococcus epidermidis and Streptococcus pneumoniae were exposed overnight to NSAIDs and the number of viable bacteria on the CLs were calculated. Cytotoxicity of NSAIDs to HCE cells was evaluated with the MTT assay. Viable counts were used to measure the adhesion of P. aeruginosa and S. epidermidis to HCE cells in the presence of the least cytotoxic NSAID. RESULTS: All NSAIDs significantly decreased bacterial colonization of CLs in a dose-dependent manner. Salicylic acid (100 mM) completely inhibited colonisation of all species tested and was the least cytotoxic. Salicylic acid also prevented adhesion of P. aeruginosa and S. epidermidis to HCE (60% and 58% inhibition at 60 mM at 2 hours). CONCLUSIONS: Salicylic acid demonstrated potential as a compound for incorporation into anti-bacterial strategies to prevent bacterial contamination of contact lenses. This study highlighted the potential for NSAIDs as anti-bacterial agents and indicates that this class of compound should be investigated for other suitable candidates.

Furanones as potential anti-bacterial coatings on biomaterials.

A major barrier to the long-term use of medical devices is development of infection. Staphylococcus epidermidis is one of the most common bacterial isolates from these infections with biofilm formation being their main virulence factor. Currently, antibiotics are used as the main form of therapy. However with the emergence of staphylococcal resistance, this form of therapy is fast becoming ineffective. In this study, the ability of a novel furanone antimicrobial compound to inhibit S. epidermidis adhesion and slime production on biomaterials was assessed. Furanones were physically adsorbed to various biomaterials and bacterial load determined using radioactivity. Slime production was assessed using a colorimetric method. Additionally, the effect of the furanone coating on material surface characteristics such as hydrophobicity and surface roughness was also investigated. The results of this study indicated that there was no significant change in the material characteristics after furanone coating. Bacterial load on all furanone-coated materials was significantly reduced (p<0.001) as was slime production (p<0.001). There is a potential for furanone-coated biomaterials to be used to reduce medical device-associated infections.

Biological performance of a novel synthetic furanone-based antimicrobial.

Infection of medical devices causes significant morbidity and mortality and considerable research effort has been directed at solving this problem. The aim of this study was to assess the biological performance of a novel furanone compound that has potential as an anti-infective coating for medical devices. This study examined in vitro leukocyte response following exposure to the antibacterial 3-(1'-bromohexyl)-5-dibromomethylene-2(5H)-furanone and assessed the tissue response following subcutaneous implantation of the furanone compound covalently bound to polystyrene (PS). Peripheral human blood was exposed to furanones in solution for 1h and flow cytometry used to analyse viability and changes in expression of surface receptors CD11b/CD18 and CD44. Flow cytometry results from propidium iodide stained cell suspensions suggested that the leukocytes were viable after exposure to furanones in whole blood. No significant difference was found in the expression of CD11b/CD18 and CD44 between the furanone exposed samples and the negative control for neutrophils suggesting that the furanones themselves do not activate these leukocytes. The positive control lipopolysaccharide significantly up-regulated CD11b/CD18 and slightly down-regulated CD44 on both PMNs and monocytes. In vivo studies of the tissue response to furanone covalently bound to PS showed that there was no significant difference in cellularity of capsules surrounding the disk and no significant increase in myeloperoxidase expression. These results demonstrate negligible acute inflammatory response to synthetic brominated antibacterial furanones. Future studies will focus on chronic responses and examination of in vivo efficacy.

The control of Staphylococcus epidermidis biofilm formation and in vivo infection rates by covalently bound furanones.

In order to overcome the continuing infection rate associated with biomaterials, the use of covalently bound furanones as an antibiofilm coating for biomaterials has been investigated. Furanones have previously been shown to inhibit growth of Gram-positive and Gram-negative bacteria. The aim of these studies were to covalently bind furanones to polymers and to test their efficacy for inhibiting biofilm formation of Staphylococcus epidermidis and in vivo infection rate. Two methods of covalent attachment of furanones were used. The first, a co-polymerisation with a styrene polymer, and second, a plasma-1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) reaction to produce furanone-coated catheters. Biofilm formation by S. epidermidis in vitro was inhibited by 89% for polystryene-furanone disks and by 78% by furanone-coated catheters (p<0.01). In an in vivo sheep model we found furanones were effective at controlling infection for up to 65 days. Furanones have potential to be used as a coating for biomaterials to control infection caused by S. epidermidis.

The causes of and cures for contact lens-induced peripheral ulcer.

PURPOSE: Contact lens-induced peripheral ulceration (CLPU) is a relatively common adverse response associated with wearing hydrogel lenses, especially on an extended wear schedule. Bacteriologic examination of lenses at the time of an event has demonstrated an association with Staphylococci spp. We sought to investigate the causes of CLPU in a rabbit model of contact lens wear. METHODS: Rabbits wore contact lenses for a period of 24 hr in the presence or absence of bacteria or in presence or absence of epithelial scratches made in the periphery of the cornea before lens wear. Bacteria tested were a strain of Staphylococcus aureus or a strain of Staphylococcus epidermidis isolated from human CLPUs. Rabbits were also challenged with S. aureus in the presence of an epithelial defect and in the absence of a contact lens. Corneas were monitored by slitlamp, histology, and microbial culture after 24 hr. RESULTS: No CLPU-like lesions were detected under the following conditions: corneal scratch plus lens wear with no bacteria; corneal scratch plus S. epidermidis colonized lenses; corneal scratch without lens wear and with S. aureus applied to eyes; no scratch plus S. aureus colonized lenses; and corneal scratch plus contact lenses colonized by dead S. aureus. CLPU-like lesions were found only when the corneas were scratched and contact lenses colonized by viable S. aureus were applied to the eye. The histology of the lesions demonstrated a frank epithelial break with underlying stromal infiltration. Only low numbers of bacteria could be cultivated from the corneas with CLPU-like lesions. The CLPU-like lesions shared many similarities with CLPU in humans. CONCLUSION: CLPU-like lesions were only produced by S. aureus and not S. epidermidis in the presence of an epithelial abrasion and contact lens. Thus, we hypothesize that to reduce the incidence of CLPU, contact lenses designed to reduce corneal interaction and repel microbial colonization should be produced.

Role and regulation of CXC-chemokines in acute experimental keratitis.

The aim of this study was to elucidate the expression of chemokines, their role and regulation in bacterial corneal infection using three bacterial strains (Pseudomonas. aeruginosa- invasive, cytotoxic and contact lens induced acute red eye strains) which have been shown to produce three distinct patterns of corneal disease in the mouse. The predominant chemokine expressed in response to all three strains was MIP-2. Prolonged expression of high levels of MIP-2 was associated with increased severity of corneal inflammation. Significantly reduced disease severity upon administration of anti-MIP-2 antibodies suggested that MIP-2 may play an important role in the pathogenesis of Pseudomonas keratitis at least in part by being a major chemoattractant for polymorphonuclear leukocytes (PMN) recruitment. Interestingly, the numbers of bacteria in eyes with neutralized MIP-2 activity did not decrease even though the severity of the disease was decreased. This implies PMNs as the major destructive factor in microbial keratitis. Further, neutralization of IL-1beta activity alone using monoclonal antibodies resulted in significant reduction of both MIP-2 and KC activity indicating that chemokine levels were regulated by IL-1beta. These studies demonstrate that the regulation of MIP-2 activity may be beneficial in reducing corneal damage during microbial keratitis in rodents and perhaps that regulation of the human homologue of MIP-2, IL-8, may be useful for controlling keratitis in humans.

Balance of pro- and anti-inflammatory cytokines correlates with outcome of acute experimental Pseudomonas aeruginosa keratitis.

The purpose of this study was to elucidate the expression of pro- and anti-inflammatory cytokines in mouse corneas infected with Pseudomonas aeruginosa. Three bacterial strains (invasive, cytotoxic, or CLARE [contact lens-induced acute red eye]) which have recently been shown to produce distinct patterns of corneal disease in the mouse were used. The left mouse (BALB/c) corneas were scarified and infected with 2 x 10(6) CFU of one of the three P. aeruginosa strains, while right eyes served as controls. Animals were examined at 1, 4, 8, 16, and 24 h with a slit lamp biomicroscope to grade the severity of infection. Following examination, eyes were collected and processed for histopathology, multiprobe RNase protection assay for cytokine mRNA, enzyme-linked immunosorbent assay to quantitate cytokine proteins, and myeloperoxidase activity to quantitate polymorphonuclear leukocytes. The kinetics of appearance and magnitude of expression of key cytokines varied significantly in the three different phenotypes of P. aeruginosa infection. The predominant cytokines expressed in response to all three phenotypes were interleukin-1 beta (IL-1 beta), IL-1Ra, and IL-6. In response to the invasive strain, which induced severe corneal inflammation, significantly lower ratios of IL-1Ra to IL-1 beta were present at all time points, whereas corneas challenged with the CLARE strain, which induced very mild inflammation, showed a high ratio of IL-1Ra to IL-1 beta. The outcome of infection in bacterial keratitis correlated with the relative induction of these pro- and anti-inflammatory cytokines, and exogenous administration of recombinant rIL-1Ra (rIL-1Ra) was able to reduce the disease severity significantly. These findings point to the therapeutic potential of rIL-1Ra protein in possible treatment strategies for bacterial keratitis.

Elucidation of the antistaphylococcal action of lactoferrin and lysozyme.

The cationic tear proteins lactoferrin and lysozyme exhibit co-operative antistaphylococcal properties. The purpose of this study was to determine the mechanism of action of this co-operation on Staphylococcus epidermidis. Following blocking of lipoteichoic acid (LTA) binding sites, the effects on binding of lactoferrin and susceptibility to lactoferrin and lysozyme were determined. The effect of lactoferrin on autolysis and LTA release was also examined. Maximal susceptibility occurred on addition of lactoferrin first followed by lysozyme. Blocking the LTA binding sites both reduced lactoferrin binding and decreased susceptibility. Autolytic activity decreased and LTA release increased in the presence of lactoferrin. These results suggest that binding of lactoferrin to LTA is important in its synergy with lysozyme and interferes with the autolysins present on the LTA. It is proposed that, on binding to the anionic LTA of S. epidermidis, the cationic protein lactoferrin decreases the negative charge, allowing greater accessibility of lysozyme to the underlying peptidoglycan.