Optimization of goblet cell density quantification methods.

The purpose of this study was to develop a standardized, accurate and efficient method for estimating conjunctival goblet cell density (GCD) via optimizing sample storage conditions and quantification methods. Conjunctival impression cytology (CIC) membranes were collected from both eyes of 32 participants and were randomized to two storage durations (2-3 weeks, 6-7 weeks) and two storage container types (microcentrifuge tube, flat histology cassette). The CIC membranes were stained and subdivided into 25 areas (5 mm × 5 mm) for imaging and the GCs were counted under 200X magnification using three different methods: (1) full CIC membrane GC count of the 25 images with cell-counting software ("full"; reference method), (2) partial membrane GC count of 9 images with cell-counting software ("partial"), and (3) manual counting of the 25 images ("manual"). In all cases, GCD was determined by dividing the GC count by the counting area. The average time required for quantification was recorded to gauge efficiency. Results showed no significant difference in GC count between the two storage durations (p = 0.745) or storage container types (p = 0.552). The median (interquartile range (IQR)) time required to quantify a CIC membrane for the full, partial, and manual methods of GC counting, was 14.8(17.6), 4.6(5.2) and 5.0 (5.0) minutes, respectively. The agreement of GCD values between the full and manual methods (bias: 0.4, 95% LOA: [-4.6, 5.5]) was stronger than that comparing the full and partial methods (bias: 0.5, 95% LOA: [-18, 17]). All together, through systematic examination of key procedural variables, an optimized method for GCD quantification within 7 weeks of sample collection was outlined. Adaption of procedures described in this paper to facilitate accurate and efficient GCD quantification may serve as a valuable step in clinical trials investigating DED pathophysiology and/or novel DED treatment strategies.

The Impact of Incubation Conditions on In Vitro Phosphatidylcholine Deposition on Contact Lens Materials.

SIGNIFICANCE: Previous in vitro measurements of contact lenses commonly investigate the impact of nonpolar tear film lipids (i.e., sterols). Polar lipids, however, are equally important stabilizing components of the tear film. This research explores and presents further knowledge about various aspects of polar lipid uptake that may impact contact lens performance. PURPOSE: This study evaluated the impact of incubation time, lipid concentration, and replenishment of an artificial tear solution (ATS) on the uptake of phosphatidylcholine (PC) onto conventional hydrogel (CH) and silicone hydrogel (SH) contact lens materials. METHODS: Four SHs and two CH lens materials (n = 4) were soaked in a complex ATS containing radioactive 14C-PC as a probe molecule. Phosphatidylcholine uptake was monitored at various incubation time points (1, 3, 7, 14, and 28 days), with different ATS lipid concentrations (0.5×, 1×, 2×) and with and without regular replenishment of the ATS. Phosphatidylcholine was extracted from the lenses, processed, and counted by a ß counter, and accumulated PC (µg/lens) was extrapolated from standard lipid calibration curves. RESULTS: All materials exhibited increasing PC deposition over time. Conventional hydrogel materials showed significantly lower PC uptake rates (P < .001) than any of the SH materials. Increasing lipid concentration in the ATS resulted in increased PC binding onto the contact lens materials (P < .001). Replenishing the ATS every other day, however, impacted the PC deposition differently, showing increased binding (P < .001) on CHs and reduced PC deposition for SH materials (P < .001). CONCLUSIONS: Length of incubation, lipid concentration in the ATS, and renewal of the incubation solution all influenced the amount of PC that sorbed onto various lens materials and therefore need to be considered when conducting future in vitro deposition studies.

Uptake and Release of a Multipurpose Solution Biocide (MAP-D) From Hydrogel and Silicone Hydrogel Contact Lenses Using a Radiolabel Methodology.

PURPOSE: The purpose of this study was to evaluate the uptake and release of radiolabelled myristamidopropyl dimethylamine (MAP-D) on reusable daily wear contact lenses (CLs) over 7 days. METHODS: Three silicone hydrogel (SH) CL materials (lotrafilcon B, balafilcon A, senofilcon A) and two conventional hydrogel (CH) materials (etafilcon A, omafilcon A) were tested. A short-term (experiment 1, N=4) and a longer-term (experiment 2, N=3) study was conducted. In experiment 1, the CLs were incubated in 2 mL of phosphate buffered solution (PBS) containing 14C MAP-D (5 µg/mL) for 8 hrs. The release of 14C MAP-D was measured at t=0.25, 0.5, 1, 2, 4, 8, and 24 hr in PBS. In experiment 2, the CLs were incubated in the 14C MAP-D solution for 8 hrs followed by a 16-hr release in PBS. This cycle was repeated daily for 7 days. At the end of both experiments, lenses were extracted to determine the total uptake of MAP-D. The radioactivity was measured using a beta scintillation counter. RESULTS: In experiment 1, all three SH lenses sorbed similar amounts of MAP-D (P=0.99), all of which were higher than the two CH materials (P<0.01). However, the CH materials released a greater amount of MAP-D than the SH materials (P<0.01). In experiment 2, the uptake of MAP-D in SH materials increased over 7 days, whereas the amount of MAP-D remained constant in the CH materials (P=0.99). Similar to experiment 1, the CH lenses released more MAP-D than SH lenses after 7 days (P<0.01). CONCLUSION: The SH materials absorbed greater amounts of MAP-D compared to CH materials. However, the CH materials released the greatest amount of MAP-D. Radioactive labelling of MAP-D offers a highly sensitive method of assessing the uptake and release profiles of biocides to CL materials.

Degradation of proteoglycan 4/lubricin by cathepsin S: Potential mechanism for diminished ocular surface lubrication in Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease affecting the lacrimal and salivary glands with hallmark clinical symptoms of dry eye and dry mouth. Recently, markedly increased cathepsin S (CTSS) activity has been observed in the tears of SS patients. Proteoglycan 4 (PRG4), also known as lubricin, is an effective boundary lubricant that is naturally present on the ocular surface. While PRG4 is susceptible to proteolytic digestion, the potential effect of CTSS on PRG4 remains unknown. The objective of this study was to assess the ability of CTSS to enzymatically degrade purified PRG4, and PRG4 naturally present in human tears, and alter ocular surface boundary lubricating properties. To assess the potential time course and dose-dependency of PRG4 digestion by CTSS, full-length recombinant human PRG4 (rhPRG4) was incubated at 37 °C with or without CTSS in an enzymatic digestion buffer. Digestion of PRG4 by CTSS was also examined within normal human tear samples, both with and without supplementation by rhPRG4. Finally, digestion of endogenous PRG4 by CTSS, and the effect of a CTSS inhibitor, was examined in SS tears on Schirmer strips. Digestion products were separated on 3-8% SDS-PAGE and visualized by protein staining and western blotting. The boundary lubricating ability of rhPRG4 samples was assessed using an in vitro human eyelid-cornea friction test. Finally, SDS-PAGE protein stain bands resulting from rhPRG4 digestion were submitted for tandem mass spectrometry analysis to confirm their identity as PRG4 and identify non-tryptic cleavage sites. CTSS digested rhPRG4 in a time and dose dependent manner. CTSS digestion of rhPRG4 at 1% (where % is the mass ratio of CTSS to rhPRG4) resulted in a time dependent decrease in the full-length, ∼460 kDa, monomeric rhPRG4 band, and an appearance of lower MW fragments. After 20 h, no full-length rhPRG4 was observed. Furthermore, with an increased relative enzyme concentration of 3%, no protein bands were observed after 2 h, indicating complete digestion of rhPRG4. Western blotting demonstrated PRG4 is present in normal human tears, and that rhPRG4, tears, and tears supplemented with rhPRG4 incubated with 3-9% CTSS demonstrated decreased intensity of high MW PRG4 bands, indicative of partial degradation by CTSS. Similarly, western blotting of PRG4 in SS tears incubated with CTSS demonstrated decreased intensity of high MW PRG4 bands, which was reversed in the presence of the CTSS inhibitor. CTSS treatment of rhPRG4 resulted in an increased friction coefficient, compared to untreated controls. Lastly, the lower MW bands were confirmed to be PRG4 fragments by tandem mass spectrometry, and 6 non-tryptic cleavage sites were identified. rhPRG4 is susceptible to proteolytic digestion by CTSS, both alone and in human tears, which results in diminished ocular surface boundary lubricating ability. Moreover, endogenous PRG4 is susceptible to proteolytic digestion by CTSS, both in normal and SS tears. Given the elevated activity of CTSS in SS tears, and the role intact PRG4 plays in ocular surface health and lubrication, degradation of PRG4 by CTSS is a potential mechanism for diminished ocular surface lubrication in SS. Collectively these results suggest that tear supplementation of PRG4 may be beneficial for SS patients.

In Vitro Effect of Lysozyme on Albumin Deposition to Hydrogel Contact Lens Materials.

SIGNIFICANCE: Albumin deposition on contact lenses could be detrimental to contact lens (CL) wear because this may increase the risk of bacterial binding and reduce comfort. Lysozyme deposition on selected lens materials would reduce albumin deposition on lenses. PURPOSE: This study aims to determine if lysozyme deposition on CLs could act as a barrier against subsequent albumin adsorption, using an in vitro model. METHODS: Six hydrogel CL materials (etafilcon A, polymacon, nelfilcon A, omafilcon A, ocufilcon B, and nesofilcon A) were evaluated. Four CLs of each type were soaked in lysozyme solution for 16 hours at 37°C. Lysozyme-coated lenses were then placed in vials with 1.5 mL of artificial tear solution containing I-labeled albumin for 16 hours at 37°C with shaking. Four uncoated lenses of each type were used as controls. Lenses soaked in radiolabeled albumin were rinsed in a phosphate-buffered saline solution, and radioactive counts were measured directly on lenses using a gamma counter. Albumin uptake on lenses was measured using a calibration curve by plotting radioactive counts versus protein concentration. RESULTS: Results are reported as mean ± SD. Lysozyme-coated etafilcon A lenses exhibited lower levels of deposited albumin than uncoated etafilcon A lenses (58 ± 12 vs. 84 ± 5 ng/lens; P < .05). There were no differences in albumin adsorption between control (uncoated) and lysozyme-coated polymacon (105 ± 10 vs. 110 ± 34 ng/lens), nelfilcon A (51 ± 7 vs. 42 ± 20 ng/lens), omafilcon A (90 ± 20 vs. 80 ± 38 ng/lens), ocufilcon B (87 ± 20 vs. 115 ± 50 ng/lens), and nesofilcon A (170 ± 29 vs. 161 ± 10 ng/lens) lens materials (P > .05). Uncoated nesofilcon A lenses deposited the highest amount of albumin when compared with other uncoated lenses (P < .05). CONCLUSIONS: This study demonstrates that lysozyme deposited onto etafilcon A resists the deposition of albumin, which may potentially be beneficial to CL wearers.

Impact of Lens Care Solutions on Protein Deposition on Soft Contact Lenses.

PURPOSE: To evaluate the effect of four contemporary lens care solutions on total protein, total lysozyme, and active lysozyme extracted from three contact lens materials. METHODS: Adapted contact lens wearers were recruited at three sites, and all subjects were randomly assigned to daily wear of either etafilcon A, galyfilcon A, or senofilcon A for 2 weeks. Four lens care solutions (Biotrue, OPTI-FREE PureMoist, RevitaLens OcuTec, and ClearCare) were used by each subject in random order with a new pair of lenses after a washout period between solutions of at least 4 days. After 2 weeks of daily wear, contact lenses were collected for analysis. Proteins were extracted from a subset of contact lenses (n = 568) and total protein, total lysozyme, and lysozyme activity were quantified using a modified Bradford assay, an enzyme-linked immunosorbent assay, and a micrococcal assay, respectively. RESULTS: Higher levels of total protein were extracted from etafilcon A when used with Biotrue compared to other solutions (p = 0.0001). There were higher levels of total lysozyme extracted from galyfilcon A lenses when used with PureMoist than with Biotrue or ClearCare (p < 0.006). Higher total lysozyme was extracted from senofilcon A when used with RevitaLens OcuTec compared to Biotrue (p = 0.002). Lower lysozyme activity was recovered from senofilcon A lenses with RevitaLens OcuTec when compared to all other care solutions (all p < 0.004). When Biotrue, PureMoist, or RevitaLens OcuTec were used, higher total lysozyme was extracted from galyfilcon A compared to senofilcon A (p < 0.01). When RevitaLens OcuTec was used, higher levels of active lysozyme were extracted from galyfilcon A compared to senofilcon A (p = 0.02). CONCLUSIONS: The ability of lens care solutions to remove protein from lenses varies depending upon the care solution composition and also the polymeric make-up of the contact lens material.

Quantification of conjunctival TNF-α in aqueous-deficient dry eye.

PURPOSE: This study aimed to quantify and compare conjunctival epithelial tumor necrosis factor (NF) α mRNA expression in Sjögren syndrome (SS), non-Sjögren syndrome aqueous-deficient dry eye (non-SS DE), and non-dry eye (NDE) control subjects. METHODS: A total of 76 subjects were recruited for this study: 25 SS (confirmed via American-European Consensus Criteria 2002), 25 non-SS DE (confirmed by symptoms and Schirmer scores ≤ 10 mm), and 26 NDE. Superior and temporal bulbar conjunctival epithelial cells were collected via impression cytology. Epithelial RNA was extracted, and TNF-α mRNA expression was quantified by real-time quantitative polymerase chain reaction. RESULTS: The expression of TNF-α mRNA was found to be significantly higher in the SS group (2.48 ± 1.79) compared to both non-SS DE (0.95 ± 1.18; p < 0.05) and NDE (0.84 ± 0.51; p < 0.05) groups. No difference in TNF-α mRNA expression was found between the non-SS DE and NDE groups (p = 0.67). CONCLUSIONS: These results demonstrate that SS-associated aqueous-deficient dry eye is associated with a significant upregulation of conjunctival epithelial TNF-α mRNA relative to both non-SS DE and control groups. The degree to which TNF-α mRNA is upregulated in SS may contribute to the severe ocular surface damage observed in these patients.

Quantification of MUCIN 1, cell surface associated and MUCIN16, cell surface associated proteins in tears and conjunctival epithelial cells collected from postmenopausal women.

PURPOSE: To quantify the expression of mucin 1, cell surface associated (MUC1) and mucin 16, cell surface associated (MUC16) proteins and messenger ribonucleic acid (mRNA) in a cohort of postmenopausal women (PMW), to explore the relationship between mucin expression, dry eye symptomology, and tear stability. METHODS: Thirty-nine healthy PMW (>50 years of age) were enrolled in this study. No specific inclusion criteria were used to define dry eye; instead, a range of subjects were recruited based on responses to the Allergan Ocular Surface Disease Index (OSDI) questionnaire and tear stability measurements as assessed by non-invasive tear breakup time (NITBUT). Tears were collected from the inferior tear meniscus using a disposable glass capillary tube, and total RNA and total protein were isolated from conjunctival epithelial cells collected via impression cytology. Expression of membrane-bound and soluble MUC1 and MUC16 were quantified with western blotting, and expression of MUC1 and MUC16 mRNA was assessed with real-time PCR. RESULTS: OSDI responses ranged from 0 to 60, and NITBUT ranged from 18.5 to 2.9 s. Only two statistically significant correlations were found: soluble MUC16 protein concentration and MUC16 mRNA expression with OSDI vision related (-0.47; p=0.01) and ocular symptom (0.39; p=0.02) subscores, respectively. Post hoc exploratory analysis on absolute expression values was performed on two subsets of subjects defined as asymptomatic (OSDI≤6, n=12) and moderate to severe symptomatic (OSDI≥20, n=12). The only significant difference between the two subgroups was a significant reduction in MUC16 mRNA expression found in the symptomatic dry eye group (1.52±1.19 versus 0.57±0.44; p=0.03). CONCLUSIONS: A broad exploration of mucin expression compared to either a sign (NITBUT) or symptoms of dry eye failed to reveal compelling evidence supporting a significant relationship, other than a potential association between MUC16 with specific symptoms. Furthermore, comparison of mucin protein and expression levels between the asymptomatic and moderate to severe symptomatic subgroups revealed only one significant difference, a reduction in MUC16 mRNA expression in the symptomatic subgroup.

Factors that influence in vitro cholesterol deposition on contact lenses.

PURPOSE: The purpose of this study was to analyze the impact that incubation time, lipid concentration, and solution replenishment have on silicone hydrogel (SiHy) and conventional hydrogel (CH) contact lens cholesterol deposition via in vitro radiochemical experiments. METHODS: Four SiHy (senofilcon A, lotrafilcon B, comfilcon A, balafilcon A) and two CH (etafilcon A and omafilcon A) contact lenses were incubated in an artificial tear solution (ATS) that contained major tear film proteins, lipids, salts, salts, and a trace amount of radioactive C-cholesterol. Lenses were incubated for various incubation times (1, 3, 7, 14, or 28 days), with three concentrations of lipid (0.5×, 1×, 2× tear film concentration) and with or without solution replenishment to assess each variable's impact on cholesterol deposition. After incubation, the lenses were extracted using 2:1 chloroform:methanol, extracts were analyzed in a beta counter and masses (micrograms per lens) were extrapolated from standard curves. RESULTS: Within the SiHy materials, balafilcon A deposited the greatest amount of cholesterol (p < 0.001) and lotrafilcon B the lowest (p < 0.001). The CH lens materials showed significantly lower uptake amounts than any of the SiHy lens materials (p < 0.001). The uptake of cholesterol ranged from 0.01 ± 0.01 µg/lens to 3.22 ± 0.34 µg/lens for all lens materials. Kinetic uptake of cholesterol was shown to be continuous throughout the 28 days of incubation without plateau (p < 0.001), and varying the lipid concentration did impact the resulting cholesterol deposition (p < 0.001). Replenishing the ATS every other day also affected cholesterol deposition throughout the experiment. Overall, the deposition pattern was 2× > replenishing > 1× > 0.5×. CONCLUSIONS: Overall, SiHy lenses deposit significantly more cholesterol than CH lens materials, and the mass of lipid deposited is dependent on the contact lens material, length of incubation, concentration of lipids in the ATS, and the replenishment of ATS.

Composition of incubation solution impacts in vitro protein uptake to silicone hydrogel contact lenses.

PURPOSE: To determine the impact of incubation solution composition on protein deposition to silicone hydrogel (SH) contact lenses using a simplistic and a complex model of the tear film. METHODS: Three SH materials--senofilcon A (SA), lotrafilcon B (LB), and balafilcon A (BA)--were incubated in two different solutions; Solution A was a simplistic augmented buffered saline solution containing a single protein, whereas Solution B was a complex artificial tear solution (ATS), containing the augmented buffered saline solution in addition to proteins, lipids, and mucins (pH=7.4). The proteins of interest (lysozyme, lactoferrin, albumin) were radiolabeled with Iodine-125 (2% protein of interest) and the accumulation of the conjugated protein to the lens materials was determined after 1, 7, 14, and 28 days of incubation. Protein deposition was measured using a gamma counter and the raw data were translated into absolute amounts (µg/lens) via extrapolation from standards. RESULTS: After 28 days, lysozyme uptake was significantly lower on BA lenses when incubated in Solution A (33.7 µg) compared to Solution B (56.2 µg), p<0.001. SA lenses deposited similar amounts of lysozyme when incubated in either Solution A (2.6 µg) or Solution B (4.1 µg), p>0.05. LB lenses also deposited similar amounts of lysozyme for both solutions (Solution A: 5.0 µg, Solution B: 4.7 µg, p>0.05). After 28 days, BA lenses accumulated approximately twice the amount of lactoferrin than the other lens materials, with 30.3 µg depositing when exposed to Solution A and 22.0 µg with Solution B. The difference between the two solutions was statistically significant (p<0.001). LB materials deposited significantly greater amounts of lactoferrin when incubated in Solution A (16.6 µg) compared to Solution B (10.3 µg), p<0.001. Similar amounts of lactoferrin were accumulated onto SA lenses regardless of incubation solution composition (Solution A: 8.2 µg, Solution B: 11.2 µg, p>0.05). After 28 days, albumin deposition onto BA lenses was significantly greater when lenses were incubated in Solution B (1.7 µg) compared to Solution A (0.9 µg), p<0.001. Similar amounts of albumin were deposited on SA lenses when incubated in either solution (0.6 µg versus 0.7 µg, p>0.05). LB lenses incubated in Solution A deposited more albumin compared to Solution B (0.9 µg versus 0.6 µg), p=0.003. DISCUSSION: Protein deposition onto SH materials varied when contact lenses were incubated in either a complex ATS compared to a single protein solution. More lysozyme accumulated onto BA lenses incubated in a complex analog of the human tear film, whereas lactoferrin deposited onto SA lenses independent of incubation solution composition. To better mimic the ex vivo environment, future studies should use more appropriate analogs of the tear film.

Impact of tear film components on lysozyme deposition to contact lenses.

PURPOSE: To investigate the impact of lactoferrin and lipids on the kinetic deposition of lysozyme on silicone and conventional hydrogel lenses, using a complex artificial tear solution (ATS). METHODS: Two silicone hydrogel lenses (AIR OPTIX AQUA; lotrafilcon B and ACUVUE OASYS; senofilcon A) and two conventional hydrogel lenses (ACUVUE 2; etafilcon A and PROCLEAR; omafilcon A) were investigated. Lenses were incubated in four different solutions: a complex ATS consisting of various salts, lipids, proteins, and mucins, an ATS without lactoferrin (ATS w/o Lac), an ATS without lipids (ATS w/o Lip), and an ATS without lactoferrin and lipids (ATS w/o Lac & Lip), each containing 2% radiolabeled (125I) lysozyme (1.9 mg/ml). After each time point (4, 12 h and 1, 2, 3, 5, 7, 14, 21, 28 days), the amount of lysozyme per lens was quantified. RESULTS: After 28 days, lotrafilcon B lenses incubated in ATS deposited significantly less lysozyme (9.7 ± 1.4 µg) than when incubated in solutions not containing lactoferrin and lipids (more than 11.8 µg) (p < 0.001). Lysozyme uptake to senofilcon A lenses was higher in ATS w/o Lip (5.3 ± 0.1 µg) compared with other solutions (less than 3.9 µg) (p < 0.001). Etafilcon A lenses deposited the most lysozyme in all four solutions compared with the rest of the lens types (p < 0.001). For etafilcon A lenses, less lysozyme was deposited when incubated in ATS w/o Lip (588.6 ± 0.4 µg) compared with the other solutions (more than 642.6 µg) (p < 0.001). Omafilcon A lenses in ATS w/o Lac accumulated significantly less lysozyme (12.8 ± 1.0 µg) compared with the other solutions (more than 14.2 µg) (p < 0.001). CONCLUSIONS: An ATS containing lactoferrin and lipids impacts lysozyme deposition on both silicone and conventional hydrogel contact lenses. When performing in vitro experiments to study protein deposition on contact lenses, more complex models should be used to better mimic the human tear film.

The impact of intermittent air exposure on lipid deposition.

PURPOSE: To analyze the impact of intermittent air exposure on the in vitro deposition of two radioactive lipids on various contact lens (CL) materials, using a custom-designed model blink cell. METHODS: Six different CL materials (balafilcon A, lotrafilcon B, comfilcon A, senofilcon A, etafilcon A, and omafilcon A) were mounted on the model blink cell pistons, which cycled the lenses in and out of a complex artificial tear solution (ATS) that contained a trace amount of C-cholesterol or C-phosphatidylcholine. For the short-term experiment, air-exposed lenses were continuously cycled in and out of the ATS for 10 h. Longer term incubations for 6 days were tested with lotrafilcon B and balafilcon A materials incubated in C-cholesterol ATS. The air-exposed CLs were cycled for 14 h then submerged for 10 h each day. For both experiments, the control lenses were submerged for the entire test period. After incubation, lenses were processed, and deposited masses were quantified. RESULTS: Exposure to air resulted in increased amounts of cholesterol deposited by 1.6 to 4.3 fold on omafilcon A, balafilcon A, comfilcon A, and senofilcon A (p ≤ 0.03) compared with submerged lenses. No differences in deposition were observed for etafilcon A and lotrafilcon B (p > 0.05). The longer term incubation of lotrafilcon B and balafilcon A showed statistically significant increases in cholesterol deposition for both air-exposed lens materials (p < 0.02) with the increase in deposition 1.8× and 2.8×, respectively. For phosphatidylcholine, all air-exposed lenses had increased masses of deposition. These deposits were statistically greater by 1.1 to 1.6 times for omafilcon A, comfilcon A, lotrafilcon B, and senofilcon A (p < 0.04), but not statistically different for etafilcon A or balafilcon A (p > 0.05). CONCLUSIONS: This study found that lipid deposition profiles are CL material dependent and that intermittent air exposure can influence the mass of lipid deposited.

The impact of tear film components on in vitro lipid uptake.

PURPOSE: To analyze the influence of various tear film components on in vitro deposition of two lipids (cholesterol and phosphatidylcholine) on three contact lens materials. METHODS: Etafilcon A, balafilcon A, and senofilcon A were incubated in four different incubation solutions for 3 or 14 days: an artificial tear solution containing lipids and proteins, a protein tear solution containing proteins and the lipid of interest, a lipid tear solution containing lipids and no proteins, and a single lipid tear solution containing the lipid of interest only. Each incubation solution contained one of the two radiolabeled lipids: C-cholesterol (C) or C-phosphatidylcholine (PC). After soaking, lenses were removed from the incubation solution, the lipids were extracted and quantified using a beta counter, and masses of lipid were calculated using standard calibration curves. RESULTS: This experiment examined several different parameters influencing lipid deposition on contact lenses, including lens material, length of incubation, and the composition of the incubation solution. Overall, lipid deposited differently on different lens materials (p < 0.0005), with the order of deposition most commonly being balafilcon > senofilcon > etafilcon. Incubation solution had a large impact on how much lipid was deposited (p < 0.00001), although cholesterol and phosphatidylcholine demonstrated different deposition patterns. Lipid deposition after 14 days of incubation was consistently greater than after 3 days (p < 0.02). CONCLUSIONS: This in vitro study demonstrates that C and PC deposition are cumulative over time and that silicone hydrogel materials deposit more lipid than group IV conventional hydrogel materials. It also clearly demonstrates that deposition of C and PC is influenced by the composition of the incubation solution and that in vitro models must use more physiologically relevant incubation solutions that mimic the natural tear film if in vitro data is to be extrapolated to the in vivo situation.

Contact lens physical properties and lipid deposition in a novel characterized artificial tear solution.

PURPOSE: To characterize various properties of a physiologically-relevant artificial tear solution (ATS) containing a range of tear film components within a complex salt solution, and to measure contact lens parameters and lipid deposition of a variety of contact lens materials after incubation in this ATS. METHODS: A complex ATS was developed that contains a range of salts, proteins, lipids, mucin, and other tear film constituents in tear-film relevant concentrations. This ATS was tested to confirm that its pH, osmolality, surface tension, and homogeneity are similar to human tears and remain so throughout the material incubation process, for up to 4 weeks. To confirm that silicone hydrogel and conventional hydrogel contact lens materials do not alter in physical characteristics beyond what is allowed by the International Organization for Standardization (ISO) 18369-2. The diameter, center thickness, and calculated base curve were measured for five different lens materials directly out of the blister pack, after a rinse in saline and then following a two week incubation in the modified ATS. To test the ATS and the effect of its composition on lipid deposition, two lens materials were incubated in the ATS and a modified version for several time points. Both ATS solutions contained trace amounts of carbon-14 cholesterol and phosphatidylcholine, such that deposition of these specific lipids could be quantified using standard methods. RESULTS: This ATS is a complex mixture that remains stable at physiologically relevant pH (7.3-7.6), osmolality (304-306 mmol/kg), surface tension (40-46 dynes/cm) and homogeneity over an incubation period of three weeks or more. The physical parameters of the lenses tested showed no changes beyond that allowed by the ISO guidelines. Incubations with the ATS found that balafilcon A lenses deposit significantly more cholesterol and phosphatidylcholine than omafilcon A lenses (p<0.05) and that removing lactoferrin and immunoglobulin G from the ATS can significantly decrease the mass of lipid deposited. CONCLUSIONS: This paper describes a novel complex artificial tear solution specially designed for in-vial incubation of contact lens materials. This solution was stable and did not adversely affect the physical parameters of the soft contact lenses incubated within it and showed that lipid deposition was responsive to changes in ATS composition.

Quantification of non-polar lipid deposits on senofilcon a contact lenses.

PURPOSE: To quantify non-polar lipids deposited on senofilcon A silicone hydrogel contact lenses (J&J Acuvue OASYS) when disinfected with a no-rub one-step hydrogen peroxide system (CIBA Vision ClearCare) and a care system preserved with Polyquad & Aldox (Alcon OPTI-FREE RepleniSH). METHODS: Thirty existing soft lens wearers symptomatic of dryness were enrolled into a 4-week prospective, randomized, bilateral eye (lens type), cross-over (care regimen), daily wear, double masked study. Subjects were refitted with senofilcon A lenses, which were replaced biweekly. During each period of wear, participants used either the peroxide or preserved system. After each period of wear, lenses were collected and lipid was extracted using 1.5 ml of a 2:1 chloroform:methanol solution for 3 h at 37 °C. Lens extracts were analyzed for non-polar lipids [cholesterol oleate (CO), cholesterol, oleic acid (OA), triolein, and OA methyl ester] using normal phase high-performance liquid chromatography. RESULTS: The total lipid (sum of CO and cholesterol) detected was 34 ± 28 µg/lens for the peroxide-based system and 22 ± 21 µg/lens for the system preserved with Polyquad and Aldox (p = 0.029). Although there was no difference between products for cholesterol (1.4 vs. 1.3 µg/lens; p = 0.50), use of a system preserved with Polyquad and Aldox resulted in significantly less deposited CO (33 ± 28 vs. 21 ± 20 µg/lens; p = 0.033). Approximately, 95% of the detectable lipid deposited on the material was CO, followed by cholesterol. OA and triolein contributed <1% of the total lipid and no OA methyl ester was found on any of the lenses. CONCLUSIONS: A care system preserved with Polyquad and Aldox removed higher amounts of CO from senofilcon A contact lenses used for 2 weeks than a peroxide-based system, in soft lens wearers who were symptomatic of dry eye.

Activity of Deposited Lysozyme on Contemporary Soft Contact Lenses Exposed to Differing Lens Care Systems.

PURPOSE: The amount of protein deposition on soft contact lenses and to what extent the proteins are denatured may have an impact on comfortable wearing times of contact lenses. The purpose of this study was to evaluate the effects of two lens care systems on total protein and the quantity and activity of lysozyme deposited on worn senofilcon A, silicone hydrogel contact lenses. PARTICIPANTS AND METHODS: Thirty symptomatic soft contact lens wearers were enrolled into a 4-week prospective, randomized, bilateral eye, daily-wear, crossover, double-masked study. Participants were fitted with biweekly senofilcon A lenses and were assigned either a polyquaternium-1 and myristamidopropyl dimethylamine-containing system (OPTI-FREE RepleniSH) or a peroxide-based system (CLEAR CARE). After each wear period, proteins were extracted from the lenses and analyzed for total protein, total lysozyme quantity and activity. RESULTS: The use of either the peroxide-based system or the polyquaternium-1 and myristamidopropyl dimethylamine-containing system resulted in no difference (P>0.05) to the amount of total protein deposited on the lenses (6.7 ± 2.8 micrograms/lens versus 7.3 ± 2.8 micrograms/lens, respectively) or to the amount of denatured lysozyme deposits (0.8 ± 0.7 versus 0.9 ± 0.7 micrograms/lens), respectively. The total amount of lysozyme deposited on the lenses was significantly lower when using the peroxide-based system (1.3 ± 0.9 micrograms/lens) compared to the polyquaternium-1 and myristamidopropyl dimethylamine-containing system (1.7 ± 1.0 micrograms/lens) (P=0.02). CONCLUSION: The inactivation of lysozyme deposited on senofilcon A lenses when disinfected with the peroxide-based or the polyquaternium-1 and myristamidopropyl dimethylamine-containing systems were neither statistically nor clinically significant and the overall amounts of denatured lysozyme recovered from the lenses were low (<1 microgram/lens).

Development of an In Vitro Blink Model for Ophthalmic Drug Delivery.

PURPOSE: The purpose of this study was to develop an advanced in vitro blink model that can be used to examine the release of a wide variety of components (for example, topical ophthalmic drugs, comfort-inducing agents) from soft contact lenses. METHODS: The model was designed using computer-aided design software and printed using a stereolithography 3D printer. The eyelid and eyeball were synthesized from polyvinyl alcohol and silicone material, respectively. Simulated tear fluid was infused through tubing attached to the eyelid using a syringe pump. With each blink cycle, the eyelid slides and flexes across the eyeball to create an artificial tear film layer. The flow-through fluid was collected using a specialized trough. Two contact lenses, etafilcon A and senofilcon A, were incubated in 2 mL of a water-soluble red dye for 24 h and then placed on the eye model (n = 3). The release of the dye was measured over 24 h using a tear flow rate of 5 µL/min. RESULTS: Approximately 25% of the fluid that flowed over the eye model was lost due to evaporation, nonspecific absorption, and residual dead volume. Senofilcon A absorbed more dye (47.6 ± 2.7 µL) than etafilcon A (22.3 ± 2.0 µL). For etafilcon A, the release of the dye followed a burst-plateau profile in the vial but was sustained in the eye model. For senofilcon A, the release of the dye was sustained in both the vial and the eye model, though more dye was released in the vial (p < 0.05). Overall, the release of the dye from the contact lenses was higher in the vial compared with the eye model (p < 0.05). CONCLUSION: The blink model developed in this study could be used to measure the release of topical ophthalmic drugs or comfort agents from contact lenses. Simulation of a blink mechanism, an artificial tear film, and nonspecific absorption in an eye model may provide better results than a simple, static vial incubation model.

Lipid deposition on contact lenses in symptomatic and asymptomatic contact lens wearers.

PURPOSE: Lipid deposition on contact lenses (CL) has traditionally been believed to reduce comfort during CL wear. The purpose of this study was to quantify lipid deposition on CL in a group of symptomatic and asymptomatic adapted CL wearers. METHODS: This was a single-masked, randomized clinical trial. Only confirmed symptomatic (comfortable lens wear time (CWT) < 8 h and a noticeable reduction in comfort over the course of the day) and asymptomatic (CWT > 10 h and minimal reduction in comfort over the course of the day) participants were recruited to participate in the study. Participants wore senofilcon A lenses in combination with a polyquaternium-based care solution (OPTI-FREE Replenish). Worn CL samples were collected on Day 14. Deposited lipid amounts from the lenses (including cholesteryl ester, cholesterol and triolein) were quantified using a liquid chromatography-mass spectrometry technique. RESULTS: Lipid deposition was significantly higher in CL extracts of asymptomatic wearers compared to the symptomatic wearers for all lipid types quantified, including cholesteryl ester (2.1 ± 0.6 vs 1.6 ± 0.5 log µg/lens), cholesterol (1.5 ± 0.3 vs 1.1 ± 0.3 log µg/lens) and triolein (0.3 ± 0.2 vs 0.1 ± 0.1 log µg/lens) (all p < 0.002). The amount of cholesteryl ester deposited was greatest (p = 0.0001), followed by cholesterol, then triolein, for both the asymptomatic and symptomatic groups (both p = 0.0001). CONCLUSION: This study demonstrated that the asymptomatic group deposited a significantly greater amount of lipid on their CL. Although lipid levels measured are considered low to trigger any observable clinical deposition, they may influence other clinical outcomes, particularly comfort.

Proteomics Analysis of Tears and Saliva From Sjogren's Syndrome Patients.

Sjogren's syndrome (SS) is characterized by dysfunctional mucous membranes and dysregulated moisture-secreting glands resulting in various symptoms, including dry mouth and dry eyes. Here, we wanted to profile and compare the tear and saliva proteomes of SS patients to healthy controls. Tear and saliva samples were collected and subjected to an isotopic dimethylation labeling shotgun proteomics workflow to identify alterations in protein levels. In tear samples, we identified 83 upregulated and 112 downregulated proteins. Pathway enrichment analysis of the changing proteins by Metascape identified leukocyte transendothelial migration, neutrophil degranulation, and post-translation protein phosphorylation in tears of SS patients. In healthy controls' tears, an enrichment for proteins related to glycolysis, amino acid metabolism and apoptotic signaling pathway were identified. In saliva, we identified 108 upregulated and 45 downregulated proteins. Altered pathways in SS patients' saliva included cornification, sensory perception to taste and neutrophil degranulation. In healthy controls' saliva, an enrichment for proteins related to JAK-STAT signaling after interleukin-12 stimulation, phagocytosis and glycolysis in senescence were identified. Dysregulated protease activity is implicated in the initiation of inflammation and immune cell recruitment in SS. We identified 20 proteases and protease inhibitors in tears and 18 in saliva which are differentially expressed between SS patients and healthy controls. Next, we quantified endogenous proteoglycan 4 (PRG4), a mucin-like glycoprotein, in tear wash and saliva samples via a bead-based immune assay. We identified decreased levels of PRG4 in SS patients' tear wash compared to normal samples. Conversely, in saliva, we found elevated levels of PRG4 concentration and visualized PRG4 expression in human parotid gland via immunohistological staining. These findings will improve our mechanistic understanding of the disease and changes in SS patients' protein expression will help identify new potential drug targets. PRG4 is among the promising targets, which we identified here, in saliva, for the first time.

Cyclobutanone analogues of beta-lactams revisited: insights into conformational requirements for inhibition of serine- and metallo-beta-lactamases.

The most important mode of bacterial resistance to beta-lactam antibiotics is the expression of beta-lactamases. New cyclobutanone analogues of penams and penems have been prepared and evaluated for inhibition of class A, B, C, and D beta-lactamases. Inhibitors which favor conformations in which the C4 carboxylate is equatorial were found to be more potent than those in which the carboxylate is axial, and molecular modeling studies with enzyme-inhibitor complexes indicate that an equatorial orientation of the carboxylate is required for binding to beta-lactamases. An X-ray structure of OXA-10 complexed with a cyclobutanone confirms that a serine hemiketal is formed in the active site and that the inhibitor adopts the exo envelope. An unsaturated penem analogue was also found to enhance the potency of meropenem against carbapenem-resistant MBL-producing strains of Chryseobacterium meningosepticum and Stenotrophomonas maltophilia. These cyclobutanones represent the first type of reversible inhibitors to show moderate (low micromolar) inhibition of both serine- and metallo-beta-lactamases and should be considered for further development into practical inhibitors.