Ann Beaton

Host-Defense Mechanism of the Ocular Surfaces

The defense of the ocular surfaces presents an unique challenge in that not only must integrity be maintained against microbial, inflammatory and physical assault, but it must be done while minimizing the risk of loss of corneal transparency. This puts severe limitations on the degree to which scarring or neovascularization can occur in the cornea secondary to any infectious, inflammatory, immunological or wound healing process. Moreover, this defense system must be equally effective under two extremes of conditions: those found in the open eye and the closed eye environments. It is our contention that these constraints have resulted in the evolution of a highly complex fail-safe defense system that utilizes distinctly different strategies in open and closed eye conditions. The extraordinary effectiveness of this system is evidenced by the fact that despite continued exposure to a microbe rich environment, the external ocular surfaces maintain a very low microbial titer and are highly resistant to breaching by all but a few pathogens. It is the intent of this review to provide a working model of this defense system as it operates under both open and closed eye conditions, to provide evidence in support of this model as well as highlight some of the many areas of uncertainty.

Cytokines, matrix metalloproteases, angiogenic and growth factors in tears of normal subjects and vernal keratoconjunctivitis patients

Background:  To detect the presence of multiple mediators and growth factors in tears of vernal keratoconjunctivitis (VKC) patients with active disease using stationary phase antibody arrays.

Identification, origins and the diurnal role of the principal serine protease inhibitors in human tear fluid

PURPOSE Previous work identified polymorphonuclear leukocyte (PMN) elastase as the major caseinolytic entity in tears collected after overnight eye closure. This study was designed to identify the principal serine protease inhibitors (serpins) in tears and to determine their function in the regulation of PMN cell proteases on eye closure. METHODS Reflex and closed eye tear samples were collected by microcapillary tube and centrifuged. After reflex and closed eye supernatants (R and C) were fractionated by HPLC, samples were subjected to casein zymography and reverse zymography. Western blots were utilized to screen tears and HPLC fractions for elastase, cathepsin G and proteinase-3 and to obtain semi-quantitative data on alpha 1-protease inhibitor (alp1), alpha 1-antichymotrypsin (alpha 1-Achy), secretory leukocyte protease inhibitor (SLPI), elafin and alpha 2-macroglobulin (alpha 2-M) as well as associated complexes and products. To confirm specificity of reactivity, samples were immunoprecipitated for a given protease or serpin and screened for the coprecipitation of interacting species. RESULTS Although R fluid contains no caseinolytic activity, it contains low levels of serpin-like activity principally in the form of SLPI (5-10 ng/microliter). Lesser amounts of alpha 2-M, alpha 1-Achy and alp1 (approximately < 1-3 ng/microliter) are also evident. C fluid is associated with very high levels of PMN cell proteases along with a approximately 5-20-fold increase in the concentrations of all of the above inhibitors. Trace levels of elafin were also detected. The concentrations of rapid reacting inhibitors exceeded that of proteases, with SLPI, alpha 1-Achy and alp1 being the principal functional entities. In atypical samples, complexes of elastase and alpha 2-M were also encountered. CONCLUSIONS SLPI, a known antimicrobial agent and an elastase and cathepsin G inhibitor, is the principal serpin in R fluid. C fluid is associated with a marked increase in the concentrations of an array of rapid reacting serpins capable of inhibiting all known PMN cell serine proteases. In the normal closed eye, the concentration of rapid reacting inhibitors always exceeds that of proteases with C fluid also containing a functional reserve of the slow reacting inhibitor alpha 2-M.

Profile of Lipid and Protein Autacoids in Diabetic Vitreous Correlates with the Progression of Diabetic Retinopathy

OBJECTIVE This study was aimed at obtaining a profile of lipids and proteins with a paracrine function in normal and diabetic vitreous and exploring whether the profile correlates with retinal pathology. RESEARCH DESIGN AND METHODS Vitreous was recovered from 47 individuals undergoing vitreoretinal surgery: 16 had nonproliferative diabetic retinopathy (NPDR), 15 had proliferative diabetic retinopathy, 7 had retinal detachments, and 9 had epiretinal membranes. Protein and lipid autacoid profiles were determined by protein arrays and mass spectrometry–based lipidomics. RESULTS Vitreous lipids included lipoxygenase (LO)- and cytochrome P450 epoxygenase (CYP)-derived eicosanoids. The most prominent LO-derived eicosanoid was 5-hydroxyeicosate traenoic acid (HETE), which demonstrated a diabetes-specific increase (P = 0.027) with the highest increase in NPDR vitreous. Vitreous also contained CYP-derived epoxyeicosatrienoic acids; their levels were higher in nondiabetic than diabetic vitreous (P < 0.05). Among inflammatory, angiogenic, and angiostatic cytokines and chemokines, only vascular endothelial growth factor (VEGF) showed a significant diabetes-specific profile (P < 0.05), although a similar trend was noted for tumor necrosis factor (TNF)-α. Soluble VEGF receptors R1 and R2 were detected in all samples with lowest VEGF-R2 levels (P < 0.05) and higher ratio of VEGF to its receptors in NPDR and PDR vitreous. CONCLUSIONS This study is the first to demonstrate diabetes-specific changes in vitreous lipid autacoids including arachidonate and docosahexanoate-derived metabolites indicating an increase in inflammatory versus anti-inflammatory lipid mediators that correlated with increased levels of inflammatory and angiogenic proteins, further supporting the notion that inflammation plays a role the pathogenesis of this disease.

Antibody protein array analysis of the tear film cytokines.

Purpose. Many bioactive proteins including cytokines are reported to increase in dry eye disease although the specific profile and concentration of inflammatory mediators varies considerably from study to study. In part, this variability results from inherent difficulties in quantifying low abundance proteins in a limited sample volume using relatively low sensitivity dot ELISA methods. Additional complexity comes with the use of pooled samples collected using a variety of techniques and intrinsic variation in the diurnal pattern of individual tear proteins. The current study describes a recent advance in the area of proteomics that has allowed the identification of dozens of low abundance proteins in human tear samples. Methods. Commercially available stationary phase antibody protein arrays were adapted to improve suitability for use in small volume biological fluid analysis with particular emphasis on tear film proteomics. Arrays were adapted to allow simultaneous screening for a panel of inflammatory cytokines in low volume tear samples collected from individual eyes. Results. A preliminary study comparing tear array results in a small population of Sjögren’s syndrome patients was conducted. The multiplex microplate array assays of cytokines in tear fluid present an unanticipated challenge due to the unique nature of tear fluid. The presence of factors that exhibit an affinity for plastic, capture antibodies and IgG and create a complex series of matrix effects profoundly impacting the reliability of dot ELISA, including with elevated levels of background reactivity and reduction in capacity to bind targeted protein. Conclusions. Preliminary results using tears collected from patients with Sjögren’s syndrome reveal methodological advantages of protein array technology and support the concept that autoimmune-mediated dry eye disease has an inflammatory component. They also emphasize the inherent difficulties one can face when interpreting the results of micro-well arrays that result from blooming effects, matrix effects, image saturation and cross-talk between capture and probe antibodies that can greatly reduce signal-to-noise and limit the ability to obtain meaningful results.

Changes in the diurnal pattern of the distribution of gelatinases and associated proteins in normal and pathological tear fluids: evidence that the PMN cell is a major source of MMP activity in tear fluid.

The matrix metalloproteases (MMP) play critical roles in modulating apoptosis, angiogenesis, cell migration, wound healing, tissue remodeling and inflammation in all connective tissues. Two extracellular MMPs, both type IV gelatinases, MMP-2 and MMP-9 (gelatinase A and B), as well as their respective inhibitors TIMP-1, TIMP-2 and TIMP-3 have been detected in the human cornea. MMP-9 is localized in the epithelium while MMP-2 is expressed preferentially by stromal keratocytes, and to a lesser extent, in the epithelium with expression of both enzymes up-regulated during wound healing.1 MMPs, principally pro-MMP-9 and associated complexes, have been detected in human tear fluid in the open eye condition. Active MMP-9 is presumed to be derived principally from the epithelium. Increased levels of enzyme and a shift in the pattern of distribution from proenzyme to the active MMP-9 species, along with the emergence of pro-MMP-2 and active MMP-2, has been observed in tear fluid in a wide range of pathological conditions. These conditions include external ocular infections, corneal wound healing secondary to LASIK, sterile corneal ulcers, dry eye syndrome, active keratoconus, ocular rosacea and sterile corneal melts.2–4 This study was designed to examine the origins and the nature of gelatinolytic species and associated proteins present in normal and pathological tear fluid during open and closed eye phases of the diurnal cycle.

Effect of external ocular surgery and mode of post-operative care on plasminogen, plasmin, angiostatins and a2-macroglobulin in tears

Purpose. To determine whether corneal surgery and the mode of post-surgical treatment influence the distribution of plasminogen, plasmin, angiostatins and a 2 -macrogobulin in tear fluid. Methods. Subjects underwent either photorefractive keratectomy (PRK), insertion of intra-stromal corneal rings (ICR), or cataract ablation followed by insertion of an intra-ocular lens (IOL). Post-surgical treatment consisted of prophylactic use of antibiotic and anti-inflammatory agents followed either by patching for 24 hours, or covering the wounded cornea with a bandage soft contact lens. Open eye tear fluid (OTF) was obtained prior to surgery and 10 minutes after patch removal or 24 hours after surgery and thereafter with the bandage lens still in place. After centrifugation, supernatants and controls were western blot analyzed using a protocol designed to allow the simultaneous semi-quantitative detection of a 2 -macroglobulin, plasminogen, plasmin, angiostatins and interleukin-8 (IL-8). Results. No obvious differences were apparent in OTF recovered from contralateral control eyes compared to the surgical eyes in individuals who underwent PRK surgery and whose eyes were covered with a bandage contact lens. In contrast, OTF samples recovered 10 minutes after patch removal from all individuals contained elevated levels of a 2 -macroglobulin and a diverse mixture of elevated levels of plasminogen/plasmin, angiostatins and possibly a plasmin-aa 1 -antiplasmin complex. All of these changes were seen, albeit to a lesser extent, in the patched control OTF samples. IL-8 could not be detected in any sample. The composition of the tear film returned to near normal on subsequent sampling 24 hours after patch removal. Conclusions. Patching results in a marked increase in the concentration of various proteins which could modulate inflammation and wound healing.

Protein Array Characterization of Bioactive Proteins Secreted By Immortalized Human Corneal Epithelium in Response to Pseudomonas Constituents

Purpose: To use protein arrays to delineate the spectrum of angiogenic bioactive protein modulators that might be secreted and up-regulated by the corneal epithelium in response to killed bacterial products. Methods: Immortalized human corneal epithelial cells were grown in culture, serum starved, and exposed to heat-killed Pseudomonas aeruginosa in a dose-dependent manner. The resultant culture medium was screened by antibody arrays for 43 proteins that can modulate angiogenesis and immune and inflammatory processes. Parallel analysis was carried out on tears recovered in the open and closed eye phases (OTF and CTF) of the diurnal cycle. Results: Array analysis reveals that the immortalized cells constitutively secrete several proteins and up-regulate the secretion of IL-6, IL-8, and GRO in response to killed bacteria. Also evident was the emergence of a strong signal for GM-CSF and moderate/weak signals for MCP-1, MMP-9, Leptin, and INFγ in a dose-dependent manner. Several of these proteins, including IL-6, IL-8, GRO, MMP-9, TIMP-1, and MCP-1, accumulate in the CTF. Other proteins are unique to tear fluid. Conclusions: Nine proteins were identified that are secreted by epithelium in response to killed bacteria that contribute to the innate and adaptive defense system through potentiating PMN and macrophage recruitment, activation, and opsonization in a cooperative manner. The vast majority of these proteins are angiogenic modulators, perhaps contributing to the imbalance between angiogenic and angiostatic processes and risk of corneal vascularization.

Characterization and origin of major high-molecular-weight tear sialoglycoproteins.

The purpose of this study was to characterize the nature and origin of the major high molecular weight soluble sialoglycoproteins in the tear fluid in open and closed eye environments.