Michelle Senchyna

Lysozyme and lipid deposition on silicone hydrogel contact lens materials.

Purpose. We sought to determine whether there were differences in lysozyme (quantity and conformation) and lipid deposition on in vivo worn conventional (etafilcon) and silicone hydrogel (balafilcon and lotrafilcon) contact lenses. Methods. After extraction, lysozyme concentration in each extract was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Lysozyme activity was determined by the rate of lysis of Micrococcus lysodeikticus cells. Lipid deposition was determined by high-performance liquid chromatography. Results. Lysozyme deposition on etafilcon lenses was significantly greater than that measured on silicone hydrogel (SH) lenses (985 &mgr;g per lens versus 10 and 3 &mgr;g per lens for balafilcon and lotrafilcon materials, respectively;P <0.001). The degree to which lysozyme was denatured was influenced by the lens material, with the lowest degree of denaturation (22%) seen on the conventional lens material, as compared with 50% for balafilcon and 80% for lotrafilcon (P <0.001). Lipid deposition was greatest on the SH materials, with up to 600 &mgr;g per lens of certain lipid classes being deposited on balafilcon, as compared with 20 &mgr;g per lens on etafilcon (P <0.001). Conclusion. The quantity and conformation of lysozyme and the quantity of lipid deposited on hydrogel contact lenses is significantly influenced by the composition of the lens material. SH contact lens materials deposit low levels of lysozyme and high levels of lipid deposition compared with ionic contact lens materials. Although SH materials deposit only small amounts of lysozyme, the degree of lysozyme denaturation that occurs is higher relative to that seen on ionic lens materials.

Quantitative and conformational characterization of lysozyme deposited on balafilcon and etafilcon contact lens materials

Purpose. To determine whether differences in lysozyme deposition and/or activity exist on worn etafilcon and balafilcon contact lenses following care with a polyquaternium-based system (PQ) or a polyhexanide-based system (PHMB). Methods. Following acid-based deposit extraction, lysozyme concentration was determined via Western blotting and lysozyme activity was determined by a micrococcyl assay. Results. Lysozyme deposition on etafilcon lenses was greater following disinfection with the PHMB-based system (1551 ± 371µg/lens vs 935 ± 271µg/lens; p < 0.001). Deposition on balafilcon lenses was not influenced by the care regimen (10 ± 3.5µg/lens vs 10 ± 5µg/lens; p = 0.89). For both materials, the percentage of denatured lysozyme was greater when they were exposed to the PHMB-based system (28 vs 21%; p = 0.05 (etafilcon) and 57 vs 40%; p = 0.04 (balafilcon)). Conclusions. The quantity and conformation of lysozyme deposited on hydrogel contact lens materials is significantly influenced by both lens material and care regimen.

Kinetics of in vitro lysozyme deposition on silicone hydrogel, PMMA, and FDA groups I, II, and IV contact lens materials.

We sought to compare the kinetics of in vitro lysozyme deposition on silicone hydrogel (SH), polymethyl methacrylate (PMMA), and FDA groups I, II, and IV contact lenses. Lenses were incubated in 125I-labeled lysozyme for time periods ranging from 1 hr to 28 days, and radioactive counts were determined. SH lenses and PMMA deposited less lysozyme than conventional hydrogel lenses (p < 0.05). Lysozyme accumulation on group IV lenses reached a maximum on the seventh day and then plateaued, whereas on groups I, II, and SH lenses, deposition continued to increase across all time periods, reiterating that kinetics of lysozyme deposition is highly material dependent.

Rewetting Drops Containing Surface Active Agents Improve the Clinical Performance of Silicone Hydrogel Contact Lenses

Purpose. The purpose of this study was to investigate the impact of using a rewetting drop (RWD) containing surface active agents (OPTI-FREE RepleniSH; Alcon, Fort Worth, TX) on the clinical performance and protein deposition when using a continuous-wear (CW) silicone hydrogel (SH) contact lens. Methods. Subjects wore lotrafilcon A SH lenses on a 30-day CW basis for two consecutive 1-month periods while inserting either 0.9% unpreserved unit-dose saline (control) or multidose OPTI-FREE® RepleniSH™ (test RWD). Subjective comfort and symptoms were assessed after 2 and 4 weeks with each product. After 1 month of wear with each product, lenses were collected and analyzed in the laboratory for total protein, total lysozyme, and percentage of denatured lysozyme. Results. Symptoms of dryness and comfort varied across the day regardless of drop type (p < 0.001) with dryness being maximal on waking, least in the middle of the day, and increased towards the evening. The test RWD provided greater comfort on insertion (p = 0.02), better visual quality (p < 0.01), and less mucous discharge on waking (p = 0.02) than the control product. Lysozyme deposition was significantly reduced after the use of the test RWD as compared to saline (0.73 ± 0.5 &mgr;g/lens vs. 1.14 ± 0.7 &mgr;g/lens; p < 0.001) as was total protein deposition (1.17 ± 0.7 &mgr;g/lens vs. 1.86 ± 0.8 &mgr;g/lens; p < 0.001). Lysozyme denaturation was also reduced with the use of the test RWD compared with the control (76 ± 10% vs. 85 ± 7%; p < 0.01). Conclusions. The use of a RWD containing surface active agents provided greater subjective satisfaction, reduced lysozyme and total protein deposition, and reduced denatured lysozyme than a RWD containing saline alone.

The dilution of benzalkonium chloride (BAK) in the tear film

The claim that benzalkonium chloride (BAK) synergistically enhances the antibiotic efficacy of gatifloxacin ophthalmic solution 0.3% (preserved with 0.005% BAK [50 μg/mL), Zymar; Allergan Inc., Irvine, Calif, USA) has been the subject of several studies. The purpose of this prospective clinical study was to test the hypothesis that BAK would be significantly diluted shortly after topical ocular administration and would thereafter have little or no effect on the enhancement of the antibiotic efficacy of commercial gatifloxacin on the ocular surface. This hypothesis was tested by investigators who measured the concentration of tear film BAK at successive time points after topical administration of commercial gatifloxacin. After subjects (N=10) received 5 separate instillations of a single 35-μL drop of gatifloxacin 0.3% ophthalmic solution in each eye, tear samples were collected at 30 sec, 1 min, 3 min, 5 min, and 20 min, with the use of graduated 5-μL glass microcapillaries. A validated high-performance liquid chromatography method was used to measure the concentration of BAK in each tear sample. The results showed rapid BAK dilution to 6.4 μg/mL, 3.2 μg/mL, 1.4 μg/mL, below the detection limit, and below the detection limit at 30 sec, 1 min, 3 min, 5 min, and 20 min after instillation of a single 35-μL drop of gatifloxacin. Because such rapid dilution reduces the concentration of BAK to near zero in minutes and does not allow the time (1 h) required for effective bacterial kill power, BAK is not expected to have a clinically significant effect on enhancement of the antimicrobial efficacy of gatifloxacin on the human ocular surface.

Tear lipocalin and lysozyme in Sjögren and non-Sjögren dry eye.

Purpose. To evaluate the concentration of tear lipocalin, lysozyme, and total protein in Sjogrens Syndrome (SS), non-Sjogrens keratoconjunctivitis sicca (KCS), and non-dry-eyed (NDE) individuals. Methods. Seventy-six subjects were recruited for this study: 25 SS subjects; 25 KCS subjects, and 26 NDE individuals. Symptoms were measured with a visual analogue scale. Tear flow was measured by the Schirmer I test without anesthesia. Tears were collected using an eye wash technique. Total tear protein was quantified using the DC Protein Assay Kit. Tear lipocalin and lysozyme were quantified via Western blotting performed on a Phast System. Results. By definition, the SS and KCS groups both had significantly lower mean Schirmer scores (5.12 ± 5.96 mm and 7.84 ± 7.35 mm) compared with the NDE group (23.83 ± 7.85 mm; p < 0.0001). There was no difference in mean Schirmer scores between SS and KCS groups (p = 0.19). The tear film of the SS group was characterized by significantly reduced (p < 0.0001) total protein and lipocalin concentrations compared with both KCS and NDE groups. No difference between the KCS and NDE groups was found in total protein (p = 0.92) or lipocalin (p = 0.19) concentration. In contrast, the concentration of tear film lysozyme was found to be statistically similar in all three groups examined. No statistically significant correlation was found in any group between mean Schirmer values compared with total protein, lipocalin or lysozyme concentration. Conclusion. Our data demonstrate a biochemical distinction between the Sjogrens group compared with both KCS and control groups, in that both tear lipocalin and total tear protein were significantly reduced. Although correlations were not found between protein measurements and tear flow, a combination of tests including Schirmer I and quantitation of tear film biomarkers may allow for the identification of SS patients without the need for invasive testing.

Molecular Structure of Interfacial Human Meibum Films

Meibum is the primary component of the tear film lipid layer. Thought to play a role in tear film stabilization, understanding the physical properties of meibum and how they change with disease will be valuable in identifying dry eye treatment targets. Grazing incidence X-ray diffraction and X-ray reflectivity were applied to meibum films at an air-water interface to identify molecular organization. At room temperature, interfacial meibum films formed two coexisting scattering phases with rectangular lattices and next-nearest neighbor tilts, similar to the Ov phase previously identified in fatty acids. The intensity of the diffraction peaks increased with compression, although the lattice spacing and molecular tilt angle remained constant. Reflectivity measurements at surface pressures of 18 mN/m and above revealed multilayers with d-spacings of 50 Å, suggesting that vertical organization rather than lateral was predominantly affected by meibum-film compression.

Impact of Time Between Collection and Collection Method on Human Tear Fluid Osmolarity

Abstract Aim: To generate data on the variability of tear osmolarity in a control (normal, non-dry eye) and symptomatic dry eye population (Ocular Surface Disease Index: OSDI ≥20). A secondary outcome is the determination of the effect that tear collection technique has on the osmolarity of the sample. Materials and methods: This was a two-phase study that recruited 20 subjects (n = 10 normal, n = 10 dry eye) to evaluate the influence of time between measurements (Phase I) and 30 subjects (n = 15 normal, n = 15 dry eye) to evaluate the influence of collection technique (Phase II). As part of Phase I, serial tear osmolarity measurements were performed on each eye; four separated by 15 min followed by four separated by 1 min, at each of three visits. Phase II compared the consecutive measurement of four in vivo tear samples to four in vitro measurements on tears collected and dispensed from a glass capillary tube. Results: During Phase I, the dry eye group had a significantly higher maximum osmolarity (334.2 ± 25.6 mOsm/L) compared to the normal group (304.0 ± 8.4 mOsm/L, p = 0.002). No significant differences were observed whether collections were performed at 15 or 1 min intervals. During Phase II, the in vivo osmolarity was equivalent to in vitro measurements from glass capillary tube samples for both the dry eye group (323.0 ± 16.7 mOsm/L versus 317.7 ± 24.8, p = 0.496), and for the normal subjects (301.2 ± 7.2 mOsm/L versus 301.9 ± 16.0 mOsm/L, p = 0.884). Conclusion: Symptomatic dry eye subjects exhibited a significantly higher tear osmolarity and variation over time than observed in normal subjects, reflecting the inherent tear film instability of dry eye disease. There was no change in the distribution of tear osmolarity measurements whether tears were collected in rapid succession or given time to equilibrate, and collection method had no impact on tear osmolarity.

Extraction Efficiency of an Extraction Buffer Used to Quantify Lysozyme Deposition on Conventional and Silicone Hydrogel Contact Lens Materials

Purpose. Extracting lysozyme from Food and Drug Administration group IV etafilcon lenses by using 0.2% trifluoroacetic acid and acetonitrile (TFA/ACN) is a well-established procedure. TFA/ACN has been the extraction buffer of choice for extracting proteins from silicone hydrogel contact lenses. The purpose of this study was to determine the efficiency of TFA/ACN in extracting lysozyme from silicone hydrogel and etafilcon lenses by using an in vitro model. Methods. ACUVUE 2, Focus NIGHT & DAY, O2 Optix, PureVision, and ACUVUE Advance lenses were incubated in simple lysozyme solution and a complex artificial tear solution consisting of multiple tear components containing lysozyme labeled with iodine 125. All the silicone hydrogel lenses were incubated for 28 days, whereas the ACUVUE 2 lenses were incubated for 7 days at 37°C with constant rotation. After the incubation period, radioactive counts were determined, and the lenses were placed in an appropriate volume of the buffer for 24 hours in darkness. The lenses were removed from the buffer, and radioactive counts were determined again. Results. Extraction efficiencies for lysozyme from the artificial tear solution were 97.2% ± 1.2% for ACUVUE 2, 64.3% ± 6.2% for Focus NIGHT & DAY, 62.5% ± 5.6% for O2 Optix, 53.5% ± 5.8% for PureVision, and 89.2% ± 3.4% for ACUVUE Advance. Results were similar for the lysozyme extracted after incubating in the simple lysozyme solution. Conclusions. TFA/ACN is extremely efficient at extracting lysozyme deposited on etafilcon lenses. However, it does not extract all the lysozyme deposited on silicone hydrogel lenses, and alternative extraction procedures should be sought.

Biophysical and Morphological Evaluation of Human Normal and Dry Eye Meibum Using Hot Stage Polarized Light Microscopy

PURPOSE To study melting characteristics and the morphology of human and mouse meibum. METHODS Hot stage cross-polarized light microscopy (HSPM) and immunohistochemical approaches were used. RESULTS Isolated human meibum, and meibum of mice (either isolated or within the meibomian ducts of mice), were found to be in liquid-crystal state at physiological temperatures. Melting of both types of meibum started at approximately 10°C and was completed at approximately 40°C. Melting curves of isolated meibum and meibum inside the meibomian ducts were multiphasic with at least two or three clearly defined phase transition temperatures, typically at approximately 12 ± 2°C (minor transition), 21 ± 3°C, and 32 ± 3°C, regardless the source of meibum. Melting was highly cooperative in nature. Samples of abnormal human meibum collected from dry eye patients with meibomian gland dysfunction often showed an increased presence of nonlipid, nonmelting, nonbirefringent, chloroform-insoluble inclusions of a protein nature. The inclusions were positively stained for cytokeratins. The presence of these inclusions was semiquantitatively characterized using a newly proposed 0 to 4 scale. In the presence of large amounts of these inclusions, melting characteristics of meibum and its structural integrity were altered. CONCLUSIONS HSPM is an effective tool that is suitable for biophysical and morphological evaluation of meibum. Morphological properties and melting characteristics of human meibum were found to be similar to those of mice. Abnormal meibum of many dry eye patients contained large quantities of nonlipid, protein-like inclusions, which were routinely absent in meibum of normal controls.