Immortalized Human Conjunctival Epithelial Cells Produce Functional Complement C3 and C4 Proteins.

PURPOSE: The aim of this study was to assess whether complement proteins C3 and C4 are produced by immortalized human conjunctival epithelial (HCjE) cells. METHODS: Supernatants and cell lysates from undifferentiated and differentiated HCjE cells were assayed for C3 and C4 by enzyme-linked immunosorbent assay. To measure complement protein function, supernatants and lysates were treated with heat-aggregated IgG, and soluble C5b-9 was measured. RESULTS: C3 was upregulated in supernatants from differentiated HCjE cells compared with undifferentiated HCjE cells (556.55 ± 91.75 vs. 56.95 ± 12.09 ng/mL, P <0.001). C4 was also increased in supernatants but to a much lesser extent (0.599 ± 0.476 vs. 0.172 ± 0.0133 ng/mL, P = 0.03). From HCjE cell lysates, total C3 production was 9.03 times higher in differentiated HCjE cells ( P <0.001), whereas total C4 remained relatively unchanged. After activation with heat-aggregated IgG, sC5b-9 could be detected from both undifferentiated and differentiated HCjE cell lysates, but not in the HCjE supernatants. CONCLUSIONS: HCjE cells produce C3 and C4 in sufficient quantities to support the formation of sC5b-9, confirming their biological activity and suggesting that HCjE cells likely produce all complement proteins C1 through C9.

Evaluation of the effects of latanoprost and benzalkonium chloride on the cell viability and nonpolar lipid profile produced by human meibomian gland epithelial cells in culture.

Purpose: The purpose of this study was to explore the effects of a PGF2α analog, latanoprost, and its preservative, benzalkonium chloride (BAK), on the cell viability and lipidomic expression of immortalized human meibomian gland epithelial cells (HMGECs). Methods: Differentiated HMGECs were exposed to latanoprost (0.05 to 50 µg/ml), BAK (0.2 to 200 µg/ml), or combined latanoprost-BAK (0.05-0.2 to 50-200 µg/ml). EP- and FP-type receptors, the cognate receptors of PGE2 and PGF2α, were inhibited, thereby sparing and isolating the function of each receptor to one condition. Cell viability was assessed by ATP quantitation, and lipid extracts were analyzed by ESI-MSMSALL with a Triple TOF 5600 Mass Spectrometer (SCIEX, Framingham, MA) using SCIEX LipidView 1.3. Results: Latanoprost and BAK were found to be lethal to HMGECs at the highest concentrations (p < 0.001 for both). The cytotoxicity of latanoprost was mediated through FP- and EP-independent mechanisms. Both latanoprost and BAK significantly modulated the lipidomic expression of several cholesteryl esters (8% and 30%, respectively) and triacylglycerols (10% and 12%, respectively). The combined latanoprost-BAK agent appeared to be no more toxic and to only negligibly alter the lipid profile relative to its individual components. Conclusions: The use of latanoprost and BAK in glaucoma may alter the viability of the meibomian glands and their lipid expression in vivo. Sublethal concentrations of BAK appear to modulate meibum lipid expression, particularly in relation to sterol biosynthesis. Non-preserved latanoprost had less cytotoxicity at lower doses and fewer lipidomic effects compared to BAK, further strengthening the argument in favor of BAK-free pharmaceutical preparations.

Prostaglandin E2 and F2α Alter Expression of Select Cholesteryl Esters and Triacylglycerols Produced by Human Meibomian Gland Epithelial Cells.

PURPOSE: PGF2α analogs are commonly used to treat glaucoma and are associated with higher rates of meibomian gland dysfunction (MGD). The purpose of this study was to evaluate the physiological effects of PGF2α and PGE2 on immortalized human meibomian gland epithelial cells (HMGECs). METHODS: HMGECs were immunostained for the 4 PGE2 receptors (EP1, EP2, EP3, and EP4) and 1 PGF2α receptor (FP) and imaged. Rosiglitazone-differentiated HMGECs were exposed to PGF2α and PGE2 (10-9 to 10-6 M) for 3 hours. Cell viability was assessed by an adenosine triphosphate-based luminescent assay, and lipid extracts were analyzed for cholesteryl esters (CEs), wax esters (WEs), and triacylglycerols (TAGs) by ESI-MSMSALL in positive ion mode by a Triple TOF 5600 Mass Spectrometer using SCIEX LipidView 1.3. RESULTS: HMGECs expressed 3 PGE2 receptors (EP1, EP2, and EP4) and the 1 PGF2α receptor (FP). Neither PGE2 nor PGF2α showed signs of cytotoxicity at any of the concentrations tested. WEs were not detected from any of the samples, but both CEs and TAGs exhibited a diverse and dynamic profile. PGE2 suppressed select CEs (CE 22:1, CE 26:0, CE 28:1, and CE 30:1). PGF2α dose dependently increased several CEs (CE 20:2, CE 20:1, CE 22:1, and CE 24:0) yet decreased others. Both prostaglandins led to nonspecific TAG remodeling. CONCLUSIONS: PGE2 and PGF2α showed minimal effect on HMGEC viability. PGF2α influences lipid expression greater than PGE2 and may do so by interfering with meibocyte differentiation. This work may provide insight into the mechanism of MGD development in patients with glaucoma treated with PGF2α analogs.

Triacylglycerol lipidome from human meibomian gland epithelial cells: Description, response to culture conditions, and perspective on function.

Preliminary work has shown that select triacylglycerols (TAGs) are upregulated in a preclinical model of MGD, suggesting that TAGs may be an important outcome variable in research involving human meibomian gland epithelial cells (HMGECs). The purpose of this study was to explore the HMGEC TAG lipidome in culture conditions known to influence differentiation. HMGECs were differentiated in DMEM/F12 with 10 ng/ml EGF, FBS (2% or 10%), and rosiglitazone (0, 20, or 50 µM) for two or five days. Following culture, lipids were extracted, processed, and directly infused into a Triple TOF 5600 mass spectrometer (SCIEX, Framingham, MA) with electrospray ionization. MS and MS/MSALL spectra were acquired in the positive ion mode and performed with the SWATH technology. Only the TAGs that were present in all 48 samples were included in the analysis. Multiple regression techniques were utilized to assess the effects of each factor (FBS, rosiglitazone, and culture duration) on each expressed TAG. The HMGEC TAG lipidome consisted of 115 TAGs with 42-62 carbons and zero to 10 double bonds. Fatty acyl chains had 14 to 26 carbons and zero to five double bonds. C18:1 (oleic acid, 25/115, 21.7%) and C16:0 (palmitic acid, 16/115, 13.9%) were the most common fatty acids. FBS, rosiglitazone, and culture duration were significant predictors for 93 TAGs (80.9%) with R2 values ranging from 0.20 to 0.77 (p < 0.05). FBS and rosiglitazone achieved significance (p < 0.05) for 80 (69.6%) and 67 TAGs (58.3%), respectively. Rosiglitazone demonstrated a selective upregulation of TAGs containing 16 or 18 carbons. Culture duration reached significance (p < 0.05) for only 36 TAGs (31.3%). When comparing the 10 most abundant C18:1-containing TAGs in meibum, FBS was a negative predictor for five TAGs (mean standardized coefficient [SC] = -0.58, p < 0.001), rosiglitazone was a positive predictor for six TAGs (mean SC = 0.41, p ≤ 0.03), and culture duration weakly influenced one TAG (SC = 0.27, p = 0.008). FBS and rosiglitazone, unlike culture duration, are powerful modulators of the TAG profile. Rosiglitazone induces changes that could be consistent with fatty acid synthesis, suggesting that quantifying the TAG lipidome could be an indirect measure of lipogenesis. Though both have been described as differentiating agents, FBS and rosiglitazone induce opposing effects on meibum-relevant TAGs. Culturing with rosiglitazone is associated with a TAG profile that is more consistent with the expected outcome of lipogenesis and with the profile observed in normal human meibum.

Saturation of cholesteryl esters produced by human meibomian gland epithelial cells after treatment with rosiglitazone.

PURPOSE: The purpose of this study was to compare the cholesteryl ester (CE) profiles expressed from human meibomian gland epithelial cells (HMGECs) in response to rosiglitazone-induced differentiation to that of normal human meibum. METHODS: HMGECs were cultured with rosiglitazone (vehicle control, 20 µM, or 50 µM) and fetal bovine serum (FBS, 2% or 10%) for 2 days or 5 days. Following culture, lipid extracts were processed and analyzed by ESI-MSMSALL in positive ion mode. CEs were identified using both LipidView 1.2 and PeakView 2.2 (SCIEX, Framingham, MA) and compared to literature reports of CEs in normal human meibum. RESULTS: There were 34 CEs with carbon number ranging from 14 to 34 detected from HMGECs. Across all conditions, HMGECs provided a CE profile that was 14.0% saturated, 60.6% monounsaturated, and 25.4% polyunsaturated. Culturing with 50 µM rosiglitazone and 2% FBS for 2 days resulted in the greatest number of upregulated saturated and monounsaturated CEs and downregulated polyunsaturated CEs. Five CEs were identified as being the most responsive to 50 µM rosiglitazone: CE 24:1, CE 28:1, CE 26:1, CE 18:1, and CE 22:1. CONCLUSION: Although differences in the CE profile exist between meibum and HMGECs, rosiglitazone promotes upregulation of highly expressed meibum-relevant CEs and shifts the saturation level toward a more meibum-like profile. The use of rosiglitazone as a differentiating agent is recommended in HMGEC research, and analysis by ESI-MSMSALL is encouraged to differentiate meibum-relevant CEs from other nonpolar distractors detected by vital stains.

Evaluation of Cell Harvesting Techniques to Optimize Lipidomic Analysis from Human Meibomian Gland Epithelial Cells in Culture.

The lipidomic analysis of immortalized human meibomian gland epithelial cells (HMGECs) has been proposed as a preclinical model to study meibomian gland dysfunction. An in vitro study was conducted to evaluate neutral lipid recovery following three harvesting techniques and to identify candidate lipid biomarkers of HMGECs. HMGECs were cultured in serum-containing media for two days to promote lipid production. Cells were either harvested by 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), harvested by 10 mM EDTA, or simultaneously harvested and extracted by 2:1 chloroform-methanol (CM). After extraction by a modified Folch technique, the nonpolar phase was processed and infused into a TripleTOF 5600 mass spectrometer (Sciex, Framingham, MA, USA) with electrospray ionization. MS and MS/MSall spectra were acquired. Nonpolar cholesteryl esters (CEs) were consistently detected in all samples, while wax esters were not. Only small differences in two out of twenty CEs were detected between harvesting methods. CM yielded less CE18:1 than the other methods but greater CE20:4 than the trypsin-EDTA method (p < 0.05 for all). Similar to human meibum, very long-chain CEs with carbon number (nc) ≥ 24 were detected in all samples and may serve as HMGEC lipid biomarkers. Further work is needed to address the absence of wax esters. Overall, the three harvesting methods are reasonably equivalent, though CM promotes much better efficiency and is recommended for higher throughput.

Effect of the Bruder moist heat eye compress on contact lens discomfort in contact lens wearers: An open-label randomized clinical trial.

The purpose of this study was to investigate the effect of the Bruder Moist Heat Compress on contact lens (CL) discomfort in subjects with contact lens-related dry eye (CLDE). This was a 4-week, single-center, three-arm, randomized, open-label clinical trial in subjects diagnosed with CLDE using the Contact Lens Dry Eye Questionnaire. Fifty-one CL wearers were randomized to one of three treatment groups: application of the Bruder Compress twice a day, Bruder Compress once a day, or warm washcloth used for ten minutes twice a day without reheating. Subject diaries were monitored for compliance and collected data on daily CL comfort upon awakening and throughout the afternoon. Clinical assessments included tear film break-up time (TBUT), lipid layer thickness (LLT), and meibomian gland evaluation. Statistical tests included a generalized linear model and one-way analysis of variance (ANOVA) to investigate treatment effect on comfortable wear time. Fifty-one subjects (98% female) completed the study. After treatment, subjects using a washcloth reported more uncomfortable contact lens wear time on average (mean = 5.1 ± 2.8 h) when compared with subjects who had used the Bruder Compress in Group 1 (mean = 2.8 ± 1.6 h) (p = 0.02). In the Bruder Compress groups, there was a significant reduction in the blockage of meibomian glands (p < 0.01). No significant difference in uncomfortable wear time was found between subjects using the Bruder Compress twice daily versus once daily (p = 0.48). Subjects using the Bruder Compress once daily had the highest rate of compliance at 90.2% (p < 0.01). No significant improvements were observed in TBUT (p = 0.76) or LLT (p = 0.78). The Bruder Moist Heat Compress resulted in a significant improvement in comfortable CL wear time in subjects with CLDE.

Tear Film and Meibomian Gland Characteristics in Adolescents.

PURPOSE: To establish normative values of ocular surface parameters in adolescents and explore factors associated with meibomian gland (MG) dropout. METHODS: Subjects between 8 and 17 years of age were enrolled in this cross-sectional study. All subjects were given dry eye and lifestyle questionnaires. Tear film assessments and meibography were performed. Statistical tests included a one-way analysis of variance to test differences in ocular surface parameters between age groups and linear correlations between clinical findings and lifestyle factors. RESULTS: Two hundred twenty-five subjects completed the study. Thirty-four subjects (15%) reported ocular discomfort, primarily itching. Tear meniscus height increased with age and was greatest in the oldest subjects (mean = 0.25 mm, P < 0.01). Across all subjects, meibography showed that 39% of the upper and 39% of the lower eyelids had MG dropout. The average MG dropout score was 0.50 ± 0.57 for the upper eyelids and was 0.67 ± 0.93 for the lower eyelids. There was no correlation between phone/tablet usage and MG dropout for either the upper (P = 0.39) or lower (P = 0.56) eyelids. CONCLUSIONS: The frequency of ocular symptoms in these adolescents was 15%. Because MG dropout is thought to increase with age, it was unexpected to observe that most subjects in this study had mild MG dropout in 1 or both eyelids. Although electronic device usage did not correlate with MG dropout in this study sample, it is still unclear what the effects of long-term digital device usage may have as the subjects age.

Short Tandem Repeat (STR) Profiles of Commonly Used Human Ocular Surface Cell Lines.

PURPOSE: The purpose of this study is to establish the short tandem repeat (STR) profiles of several human cell lines commonly used in ocular surface research. MATERIALS AND METHODS: Independently DNA was extracted from multiple passages of three human corneal epithelial cell lines, two human conjunctival epithelial cell lines and one meibomian gland cell line, from different laboratories actively involved in ocular surface research. The samples were then subjected to STR analysis on a fee-for-service basis in an academic setting and the data compared against that in available databases. RESULTS: The STR profiles for the human corneal epithelial cells were different among the three cell lines studied and for each line the profiles were identical across the samples provided by three laboratories. Profiles for the human conjunctival epithelial cells were different among the two cell lines studied. Profiles for the meibomian gland cell line were identical across the samples provided by three laboratories. No samples were contaminated by elements of other cell lines such as HeLa. CONCLUSIONS: This comprehensive study provides verification of STR profiles for commonly used human ocular surface cell lines that can now be used as a reference by others in the field to authenticate the cell lines in use in their own laboratories.

Relation Between Dietary Essential Fatty Acid Intake and Dry Eye Disease and Meibomian Gland Dysfunction in Postmenopausal Women.

PURPOSE: To evaluate the relationship between omega-3 (n-3) and omega-6 (n-6) fatty acids with dry eye disease (DED) and meibomian gland dysfunction (MGD). DESIGN: Cross-sectional study. METHODS: Postmenopausal women (n = 439) underwent a clinical evaluation and completed the Vio Food Frequency Questionnaire to estimate their dietary intake of n-3s and n-6s. Subjects were categorized into 2 binary classifications based on whether or not they had (1) DED and (2) MGD. Mean intake of dietary fatty acids was compared with 2-sample t tests. Univariate logistic regression models were used to estimate the odds ratios for each condition associated with each quintile of n-3s, n-6s, and n-6:n-3 ratios. RESULTS: For DED vs non-DED, there were no significant differences in n-3 intake (1.95 ± 1.47 g vs 1.92 ± 1.24 g, P = .86), n-6 intake (15.58 ± 11.56 g vs 15.44 ± 10.61 g, P = .91), and n-6:n-3 (8.30 ± 2.57 vs 8.30 ± 2.57, P = .99). For MGD vs non-MGD, there were no significant differences in n-3 intake (1.87 ± 1.35 vs 1.96 ± 1.39, P = .61), n-6 intake (15.26 ± 11.85 vs 15.62 ± 10.93, P = .80), and n-6:n-3 (8.35 ± 2.94 vs 8.28 ± 2.42, P = .84). The odds ratios (OR) for DED did not differ significantly from 1.0 for n-3, n-6, or n-6:n-3. High n-3 consumption (OR = 0.22 [0.06-0.78]) and moderate n-6 consumption (OR = 0.37 [0.15-0.91]) were associated with a decreased frequency of MGD. CONCLUSIONS: Dietary consumption of n-3s and n-6s showed no association with DED, but high n-3 consumption and moderate n-6 consumption were protective against MGD in this large sample of postmenopausal women.