Carol Ann Morris

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.

The effect of eye closure on protein and complement deposition on Group IV hydrogel contact lenses: relationship to tear flow dynamics.

PURPOSE This study was designed to determine the effect of overnight eye closure on the rate and composition of protein deposition on high water content ionic matrix soft contact lenses (Group IV SCLs) and to extrapolate from this data information on the probable change in the rate of reflex-type tear secretion associated with eye closure. METHODS Group IV SCLs were temporally sampled after equivalent periods of wear under closed eye (C) or open eye (O) conditions. Lenses were rinsed in saline and the majority of the tightly bound protein extracted at 90 degrees C in 40% urea, containing 1% SDS, 1 mM DTT, 100 mM Tris-HCl (pH 8.00). Residual protein was determined by Coomassie staining of the extracted lenses and densitometric analysis. Extracted protein was quantitated and separated by SDS-PAGE. Gels were either stained with Coomassie blue or reversibly stained with imidazole-zinc and blotted. Blots were PAS stained, or lectin and antibody probed for glycoproteins, secretory IgA (sIgA), IgG, lysozyme and complement C3. Laboratory simulated deposition studies were carried out on unworn lenses exposed to HPLC purified lysozyme. RESULTS The protein in the saline rinse, to a large degree mirrored the composition of tear fluid in which the lens had been residing (O or C). This would suggest that the saline wash consists of residual tear fluid and loosely adherent protein. In contrast, the urea extracts were highly homogeneous consisting primarily of lysozyme and to lesser extent lysozyme dimer. This supports the contention that the Group IV SCL functions in the eye much as cationic exchange resin selectively absorbing lysozyme. C extracts also proved relatively enriched in trace amounts of sIgA, IgG and complement C3 and its breakdown products. High levels of C3 and C3 breakdown products were specifically recovered only in the C worn lens extracts from a subject experiencing unilateral contact lens associated corneal infiltrates from the affected eye. In all subjects, markedly less protein (lysozyme) was recovered in urea extracts of lenses exposed to 7-8 h of closed eye as compared to open eye wear (0.20 +/- .08 versus 0.79 +/- .15 mg/lens (n = 6)). Temporal studies further revealed that deposition was linearly related to duration of wear during the initial phase of conditioning film formation giving rise to rate constants for lysozyme deposition of 2.2 +/- 0.29 (n = 5) and 0.20 +/- 0.06 microgram/min (n = 4) under open and closed eye conditions respectively. With further wear, deposition eventually reached a steady state. Under laboratory conditions, lysozyme was much rapidly and quantitatively removed from solution in a manner following a hyperbolic plot. This suggests that during the initial phase of deposition the rate of deposition is limited by the capacity of the tear fluid to deliver lysozyme to the lens surface under these two extremes of conditions. CONCLUSIONS Eye closure profoundly affects the rate of lysozyme deposition on Group IV hydrogels and the composition of minor biofilm constituents in a manner that could affect biocompatibility. Findings support the contention that eye closure results in a > 90% reduction in the rate of reflex-type tear secretion.

Mass spectrometric techniques applied to the analysis of human tears: a focus on the peptide and protein constituents.

Mass spectrometry has been widely used for the analysis of various biomolecules in complex samples, and in particular, has been established as a sensitive tool for the detection and identification of protein components in biological fluids. Traditionally, proteins have been visualized using gel electrophoresis and identified by peptide mass fingerprinting1–3. Although this technique aims to detect all proteins present in a given sample, proteins expressed at low levels, hydrophobic proteins, high mass proteins, and low molecular weight peptides are not fully represented4.

Vitronectin: Possible contribution to the closed-eye external host-defense mechanism

Eye closure causes a shift in the preocular tear film, from a reflex tear-rich layer which is in dynamic equilibrium to a secretory IgA-rich layer which is stagnant in nature. This is accompanied by complement conversion and plasminogen activation, followed by polymorphonuclear (PMN) cell recruitment. It is suggested that this shift to a secretory IgA and PMN cell-rich layer serves to protect the ocular surfaces from trapped microorganisms. The mechanisms whereby autologous damage is avoided are uncertain. In other tissues, vitronectin (VN) may be an inhibitor of complement and plasmin induced autolytic damage and a potentiator of microbial phagocytosis. Its presence in the external ocular environment is unknown. To screen for VN, normal human reflex (R), open-eye (O) and closed-eye (C) tear samples were collected, separated by SDS PAGE, and immunoblot probed. Detection was carried out using monoclonal antibodies (MAbs) raised against human VN, coupled to an avidin biotin conjugate-horseradish peroxidase amplification system. Quantitative analysis was carried out using a sandwich ELISA assay. Bovine corneas were sectioned and immunohistochemically stained with MAbs to bovine VN. Results revealed that in going from R to O to C tear fluid there is a marked progressive increase in VN (0.08, 0.75, 3.65 μ/ml). This is accompanied by a shift from the intact 75kDa molecule to the 65kDa breakdown product which is still biologically active, with further degradation occasionally encountered. Immunohistochemical staining of bovine cornea revealed that VN is localized in the corneal epithelium and stromal keratocytes. These findings are compatible with either a local or serological origin for VN, and support the contention that VN may be a component in the external closed-eye host-defense system.

The ocular surface, the tear film, and the wettability of contact lenses.

The tear film is the interface between the ocular surface and the external environment and, as such, plays several important roles.1 (i) It forms a refracting thin film that smooths out the irregular corneal surface topography. (ii) It maintains an extracellular environment for the epithelial cells of the cornea and the conjunctiva that is fairly constant in terms of pH, oxygen and carbon dioxide levels, and nutrient and growth factor concentrations. (iii) Tears dilute and wash away noxious stimuli, including bacteria, which are also combated by an elaborate and effective antibacterial system. (iv) The tear film changes its composition in response to physiological stimuli.

In vitro adsorption of tear proteins to hydroxyethyl methacrylate-based contact lens materials.

Objectives: Investigations of polymer interactions in single protein solutions is a necessary step in the elucidation of in vivo early binding events during protein deposition on hydroxyethyl methacrylate-based contact lens materials. Quantity and tenacity of binding of significant tear components to groups I and IV contact lenses was assessed. Competitive binding by these components was also examined. Methods: Adsorption on FDA groups I and IV hydrogel lenses was monitored using 125I-labeled protein. Lenses were incubated in increasing concentrations of radiolabeled single species proteins in solution. For competition experiments, concentration of each radiolabeled protein was held constant and the adsorption/sorption challenged with increasing concentrations of nonlabeled proteins. Lenses were soaked in phosphate-buffered saline to determine desorption. Results: Group IV lenses bound large amounts of lysozyme, whereas group I lenses bound highest amounts of albumin. Albumin binding to both lens types was relatively strong and could not be competed from binding by other proteins lysozyme, lactoferrin, and mucin. Mucin at high concentrations tended to positively cooperate with the binding of lactoferrin and albumin to all lenses. Conclusions: Binding of proteins to hydroxyethyl methacrylate-based hydrogel lens surfaces is affected by charge and polymer components, and perhaps manufacturing processes. Albumin binds strongly to lens surfaces, and this may play an adverse role during contact lens wear.

Structural Analysis of Secreted Ocular Mucins in Canine Dry Eye

The tear film is vital for the normal function of the ocular surface, influencing transparency, optical quality, and defense against the external environment. A functional understanding of the tear film depends on a knowledge of its components and their interactions. Recent data indicate the thickness of the trilaminar tear film to be 35–40 μm, the majority (~30 μm) contributed by a mucous gel situated adjacent to the epithelial surface.1–3 This gel contains mainly secreted mucins, which interact with a variety of other components, including a number of highly glycosylated components of the ocular surface glycocalyx.4–7

Hydrogel lens wettability and deposition in vivo

Background: Wearing a hydrogel contact lens disrupts tear film stability, resulting in a low non‐invasive surface drying time (NISDT) on the lens front surface. Reduced wettability causes deposit formation, reducing the quality of vision and lens comfort. This investigation determined the effect of commercially available HEMA‐based materials on pre‐lens tear film stability and deposition.

Vitronectin in Human Tears — Protection Against Closed Eye Induced Inflammatory Damage

Recent studies have shown that eye closure is associated with a shift in the pre-ocular tear film from a reflex fluid-rich layer in dynamic equilibrium to a secretory IgA-rich layer which is stagnant in nature.1,2,3,4,5 This is accompanied by plasminogen and complement activation and build-up of serum albumin, followed by the recruitment of PMN cells into the tear film.1–5 All of these changes are indicative of a state of sub-clinical inflammation.