Pollen enzymes degrade human tear fluid and conjunctival cells: an approach to understanding seasonal non-allergic conjunctivitis.

BACKGROUND: During pollen seasons, allergy-like symptoms can be observed in proven non-allergy sufferers. Pollen enzymes are thought to be responsible for conjunctival irritation. We investigated the influence of the well-known aggressive pollen species hazelnut (Corylus avellana) and birch pollen (Betula pendula) on both human tear fluid and conjunctival cell cultures. This study is an approach to seasonal non-allergic conjunctivitis (SNAC) syndrome. METHODS: Zymography was carried out in order to investigate the proteolytic activity of the pollen. Thereafter, human tear fluid was incubated with pollen extract, and the results were studied by polyacrylamide gel electrophoresis. In addition, cultivated conjunctival cells (CHANG cells) were incubated with pollen extracts. Cytomorphological changes were analyzed using the CASY1 Cell Counter. Cell viability was quantified via MTS assay. The viability of the cells which were incubated with pollen extract was compared to the viability of control cells. RESULTS: Pollen proteases destroy tear fluid proteins, as observed by polyacrylamide gel electrophoresis. The treatment of CHANG cells with pollen extract induced a statistically significant decrease in cell viability, depending on the pollen extract concentration and the incubation period. CONCLUSION: Evidence of the destruction of tear fluid proteins and damage to human conjunctival cells by pollen proteases explains conjunctival irritation in proven non-allergic people during the pollen season. One reason why not all people are affected by SNAC syndrome to the same extent could be differences in the concentrations of antiproteases present on the ocular surface.

Positive influence of hyaluronate on cell vitality of human conjunctival cells after alkali injuries.

BACKGROUND: To analyse the influence of hyaluronate (HA) on the cell vitality of human conjunctival cells after chemical injuries from NaOH or alkali-containing household cleaners. METHODS: Human conjunctival cells (Chang cells) were incubated either with NaOH solutions or dilutions of household cleaners. HA (0.025%) was added to the cell culture medium of part of the cells subsequent to chemical injuring. After 24 h the cell vitality of the HA-incubated cells, determined by MTS assay (Cell Titer 96(R) Aqueous One Solution Cell Proliferation Assay), was compared to the control cells without HA addition. RESULTS: The addition of HA to the cell culture medium subsequent to chemical injuring resulted in a significantly lower decrease in cell vitality (mean difference: 11.79; p = 0.00; CI = 98%). CONCLUSION: Because HA attenuates the decrease in cell vitality of alkali-damaged human conjunctival cells, formulations containing HA may be recommended for the treatment of chemical injuries to the anterior part of the eye.

Iodide protection from UVB irradiation-induced degradation of hyaluronate and against UVB-damage of human conjunctival fibroblasts.

BACKGROUND: To determine whether iodide protects from UVB irradiation-induced destruction of hyaluronate and against UVB injury of cultured human conjunctival fibroblasts. METHODS: Hyaluronate and primary cultured human conjunctival fibroblasts were incubated with various concentrations of iodide and then exposed to UV light irradiation of 312 nm. Hyaluronate destruction was determined by viscosity measurements. Cell viability was assessed with MTT assay. RESULTS: Iodide protects hyaluronate from UVB light-induced degradation of this macromolecule in a concentration-dependent manner. Incubation of human conjunctival fibroblasts with iodide inhibited cells from damage by UVB light. CONCLUSION: Iodide protects hyaluronate, a component of tear fluid and tissues of the anterior part of the eye, against UVB light-induced degradation. Also, injury of human conjunctival cells can be prevented by incubation with iodide before UVB irradiation. The mechanism of protection is likely to include an antioxidative reaction. To support the natural defence mechanisms of the eyes, the administration of an antioxidant such as iodide to artificial tears, for example, may help to prevent the damage of the eye provoked by oxidative stress.

The effect of sample treatment on separation profiles of tear fluid proteins: qualitative and semi-quantitative protein determination by an automated analysis system.

PURPOSE: Qualitative and quantitative determination of tear fluid components is of increasing interest in ophthalmology. Until now, for diagnosis and course control of some diseases of the anterior parts of the eye, different methods for tear fluid protein analysis are available. Results can be obtained by polyacrylamide gel electrophoresis (PAGE), immunochemistry, and high-performance liquid chromatography (HPLC). A new method for protein separation, identification and semi-quantitative determination on a chip-based micro-fluidic technique is used for the first time to investigate tear fluids. METHODS: Normal human reflex tears were obtained by stimulation with China mint oil and collected using glass capillary tubes. A lab-on-a-chip technology (developed by Agilent Technologies, Waldbronn, Germany, in co-operation with Caliper Technologies, Mountain View, California, USA) was used for separation and semi-quantitative determination of tear proteins. Tear fluid was separated on the Agilent 2100 Bioanalyzer in combination with the Protein 200 LabChip kit and the dedicated protein assay software. Time and temperature of the incubation with sample buffer were varied, and the influence of these parameters on protein separation profiles was studied. Tear proteins were also analysed by PAGE, and the results obtained by both methods were compared. RESULTS: The different proteins of tear fluid can be separated by the Agilent 2100 Bioanalyzer method in very short time. By this method, the molecular weight as well as the concentration of proteins can be determined. Data are automatically stored in digital format and can be retrieved and shared. Results of this technology were comparable with the protein pattern obtained by PAGE. It was confirmed by both methods that, depending on incubation time of tear fluid with sample buffer and on temperature, different protein pattern can be obtained from the tears of one specimen. CONCLUSION: Tear proteins - in contrast to serum or aqueous humour proteins - are very sensitive to changes in sample buffer temperature as well as incubation time with buffer. To obtain comparable results for tear fluid proteins, the sample buffer applied and the incubation time and temperature must be observed carefully. This can be demonstrated by both the new Agilent 2100 Bioanalyzer method and PAGE. These results are of importance when comparing tear fluid protein pattern for the diagnosis and course control of dry-eye syndrome and of other diseases of the anterior part of the eye.