On the Current Care Situation and Treatment of Ocular Mucous Membrane Pemphigoid in Germany. / Zur Versorgungssituation und konservativen Therapie des okulären vernarbenden Schleimhautpemphigoids in Deutschland.

BACKGROUND: Ocular involvement in mucous membrane pemphigoid (MMP) is relatively rare, with a prevalence of 25 cases per million population, equating to approx. 2,100 patients throughout Germany. Diagnosis can be difficult - especially in cases of isolated ocular involvement - and treatment can be complex and lengthy. Immunosuppressants or immunomodulatory drugs are often used. Due to the complexity of diagnosis and treatment, MMP patients are usually referred to specialized centers. The aim of this project was to evaluate the current care situation of patients with ocular MMP in Germany. METHODS: A paper-based survey was designed and sent to all university eye clinics and other specialized centers in Germany in April 2020. The survey asked about the existence of a specialized outpatient service, the total annual number of patients with MMP, the annual number of newly diagnosed patients, any interdisciplinary collaboration for diagnostic or therapeutic purposes, as well as the local and systemic therapy used. RESULTS: Of a total of 44 clinics, 28 (64%) responded, reporting a total average of 27 ± 42 (0 - 200) patients and 3.6 ± 2.2 (0 - 10) new cases per year. This corresponds to a total of 741 patients. Only nine (32%) of the responding clinics offer specialized MMP clinics. 93% of the centers collaborate with the local dermatology department. 79% perform serological and histological diagnostics in-house. About half of the centers (n = 16) apply a standardized treatment regime. Systemic glucocorticoids (66.7%) are most commonly used, followed by mycophenolate mofetil and dapsone (57.1%), rituximab (33.3%), azathioprine and cyclophosphamide (28.6%), as well as methotrexate (19.0%). The least frequently used treatment is intravenous immunoglobulin (14.3%). CONCLUSION: This survey of German ophthalmology departments obtained data from about one third of the estimated total cohort of all patients with MMP in Germany. These are presumed to be exclusively patients with at least one ocular involvement. The complex care of these patients is usually provided in collaboration with a dermatologist and with the use of systemic anti-inflammatory medication. Currently, an ophthalmological MMP register is being established to better record the epidemiology and care situation of this rare disease in Germany and to improve it in the long term.

The role of preterm birth, retinopathy of prematurity and perinatal factors on corneal aberrations in adulthood: Results from the Gutenberg prematurity eye study.

INTRODUCTION: Prematurity and retinopathy of prematurity (ROP) are associated with altered corneal shape and reduced visual acuity in childhood, but their long-term effects on corneal shape in later life are still unclear. This study evaluated whether prematurity and related perinatal factors are associated with corneal aberrations in adulthood. METHODS: The Gutenberg Prematurity Eye Study (GPES) is a cohort study using Scheimpflug imaging of the cornea. Associations were assessed between corneal Zernike aberrations and gestational age (GA), birth weight (BW), BW percentile, ROP occurrence, ROP treatment and other perinatal factors using univariate and multivariable linear regression analyses. RESULTS: This study involved 444 eyes of 256 individuals born preterm (aged 28.1 ± 8.4 years, 146 females) and 231 eyes of 132 individuals born full-term (aged 29.8 ± 8.9 years, 77 females). Multivariable analyses revealed an association between corneal higher-order aberrations and lower birth weight percentile (B = -0.001, p < 0.001) as well as ROP treatment (B = 0.120, p = 0.03). Corneal lower-order aberrations were also associated with lower birth weight percentile (B = -0.004; p = 0.001) and ROP treatment (B = 0.838, p = 0.01) but not with ROP occurrence. Increased corneal aberrations were correlated with lower visual acuity and the spherical equivalent refractive error. CONCLUSIONS: Perinatal factors, particularly low birth weight percentile and ROP treatment lead to a more irregular corneal shape in adulthood, thereby reducing optical image quality and potentially contributing to reduced visual acuity and altered refractive error.

Relaxin 2 fails to lower intraocular pressure and to dilate retinal vessels in rats.

PURPOSE: Recently, the vasodilator relaxin 2 has been introduced as a treatment for acute heart failure. However, its role on vessels of the eye and intraocular pressure (IOP) remains unclear though it has been hypothesized to induce a decrease IOP after intramuscular injection in humans. We aimed to test whether the hormone relaxin 2 lowers IOP and dilates retinal vessels in animals. METHODS: The IOP of female Sprague-Dawley rats before and after application of relaxin 2 was measured using an Icare Tonolab device calibrated for rats. Recombinant human relaxin 2 in phosphate-buffered saline with 0.1% bovine serum albumin was either applied as eye drops (1000, 2000 or 3000 ng/ml), injected intravitreally (500 ng/ml) or intravenously (13.3 µg/kg body weight). Retinal vessel thickness was monitored using infrared fundus images compiled with optical coherence tomography (Heidelberg Engineering) before and several time points after application of relaxin 2. RESULTS: Neither topical nor intravitreous or intravenous application of relaxin 2 lowered the IOP or changed the arterial or venous vessel diameter after 1 or 3 h after application. DISCUSSION: Now that relaxin 2 is more easily available, the hormone came again into focus as a potential glaucoma therapeutic. However, our study in rats could not support the hypothesis that relaxin 2 lowers IOP or dilates retinal vessels.

The human meibomian gland epithelial cell line as a model to study meibomian gland dysfunction.

The meibomian gland dysfunction (MGD) is the leading cause of dry eye disease (DED) throughout the world. The investigation of MGD lacks suitable in vivo and in vitro models. In 2010 a human meibomian gland epithelial cell line (HMGEC) was established, so far the only available meibomian gland cell line. The characterization of HMGEC is of major importance to clarify its suitability for studying the meibomian gland (patho)physiology in vitro. The current culture protocol and new concepts of HMGEC culture will be compared. Hormones are believed to be a key factor in meibomian gland dysfunction thus HMGEC responsiveness to hormone stimulation is crucial to elucidate the hormonal influence on the meibomian gland. This review will summarize current findings about HMGEC and discuss its role in the meibomian gland dysfunction research.

In vitro effects of sex hormones in human meibomian gland epithelial cells.

Meibomian gland dysfunction (MGD) is considered the most common cause of dry eye disease (DED). Sex hormones seem to play a role in the pathogenesis of MGD although their involvement is not completely understood. Therefore, in the present study we evaluated the effect of dihydrotestosteron (DHT) and estradiol (ß-Est) on an immortalized human meibomian gland epithelial cell line (HMGEC). Protein expression of sex hormone receptors in HMGEC was investigated by western blot. Ultrastructural morphology, Sudan III lipid staining, cell proliferation as well as vitality assays were performed. Furthermore, expression of MGD-associated markers for keratinization (hornerin, involucrin and CK6), proliferation (CK5 and CK14) and lipid synthesis (fatty acid synthase and stearoyl-CoA desaturase) were analyzed by real time RT-PCR. Western blot revealed presence of androgen receptor (AR), estrogen receptors α and -ß (ERα, ERß) and progesterone receptor (PR) in HMGEC. PR, ERα and ERß expression was significantly induced under cultivation with serum, whereas sex hormones stimulation showed no further effect on protein expression of PR, ERα and ERß. Our results showed no impact of MGD-associated sex hormones to cellular morphology and lipid accumulation in HMGEC. Cell proliferation was slightly induced through application of sex hormones and supplementation of calcium. However, both sex hormones and calcium altered gene expression of MGD-associated markers. Especially keratinization genes hornerin (HRNR) and cornulin (COR) were induced after application of sex hormones and calcium in serum-free cultivated HMGEC. This may promote keratinization processes that are associated with MGD. Further investigations are necessary to analyze the (hyper)keratinization processes that occur during MGD and using HMGEC as an in vitro model.

In vitro effects of docosahexaenoic and eicosapentaenoic acid on human meibomian gland epithelial cells.

To investigate the effect of ω-3 fatty acids on human meibomian gland epithelial cells (HMGECs, cell line) in vitro. HMGECs were stimulated with docosahexaenoic acid (DHA) or combinations with eicosapentaenoic acid (EPA) and acetyl sialic acid (ASA). Sudan III fat staining, viability and proliferation assays, electric cell-substrate impedance sensing, real-time PCR for gene expression of cyclooxygenase-2 and 15-lipoxygenase and ELISAs for resolvin D1 (RvD1), IFNγ, TNFα and IL-6 were applied. Lipid droplet accumulation and viability was increased by 100 µM DHA in the presence or absence of EPA in serum cultured HMGECs. In contrast, HMGECs cultured with DHA and EPA under serum-free conditions showed minimal lipid accumulation, decreased proliferation and viability. Normalized impedance was significantly reduced in serum-free cultured HMGECs when stimulated with DHA and EPA. HMGECs cultured in serum containing medium showed increased normalized impedance under DHA and EPA stimulation compared to DHA or EPA alone or controls. IL-6 and IFNγ were downregulated in HMGECs treated for 72 h with DHA and EPA. In general, TNFα, IFNγ and IL-6 levels were decreased after 72 h compared to 24 h in serum containing medium with or without DHA or EPA. The concentration of RvD1 was elevated 2-fold after DHA treatment. Cyclooxygenase-2 gene expression decreased compared to controls during DHA stimulation after 72 h. Treatment with DHA and ASA revealed a decreased 15-lipoxygenase gene expression which was reduced after three days of DHA incubation. DHA and EPA supplementation affected HMGECs in vitro and supported anti-inflammatory effects by influencing cytokine levels, decreasing COX-2 expression and increasing the production of RvD1.

A lower birth weight percentile is associated with central corneal thickness thinning: Results from the Gutenberg Prematurity Eye Study (GPES).

PURPOSE: Prematurity, prenatal growth restriction, and retinopathy of prematurity (ROP) are associated with altered ocular geometry, such as a steeper corneal shape in childhood, but it is unclear whether perinatal history affects corneal thickness development, so this study investigated whether corneal thickness in adulthood is affected by perinatal history. MARTERIALS AND METHODS: The Gutenberg Prematurity Eye Study (GPES) is a retrospective cohort study with a prospective ophthalmologic examination in Germany. The corneal thickness was measured by Scheimpflug imaging (Pentacam HR, Oculus Optikgeräte GmbH, Wetzlar, Germany), and the relationship between perinatal parameters respective birth weight percentile and corneal thickness at different locations was assessed using uni- and multivariable linear regression models. Covariates included age, sex, mean corneal radius, white-to-white distance, gestational age, birth weight percentile, ROP occurrence, and treatment. The main outcome measures were corneal thickness at the apex, the pupil center, and the corneal periphery. RESULTS: The corneal thickness was measured in 390 participants (754 eyes, mean age 29.7+/-8.7 years, 224 females). In multivariable analyses, a lower birth weight percentile was associated with a lower corneal thickness at the apex (B = 0.20, p = 0.003) and the pupil (B = 0.19, p = 0.007). These effects diminished towards the corneal periphery and were not observed beyond the 4-mm diameter circle around the thinnest corneal position. Neither gestational age, ROP occurrence, or ROP treatment affected the corneal thickness. CONCLUSION: A lower birth weight percentile in subjects born preterm as a proxy for restricted fetal growth is associated with corneal thickness thinning in adults aged 18 to 52 years, indicating that corneal thickness development, particularly in the corneal center, may originate in the fetal stage.

Fresh and cryopreserved amniotic membrane secrete the trefoil factor family peptide 3 that is well known to promote wound healing.

Amniotic membrane (AM) is often used for the treatment of ocular surface ulcerations and other corneal defects. Trefoil factor family (TFF) peptide 3 is produced by conjunctival goblet cells, participates in tear film physiology and has also been shown to be involved in ocular surface restitution after corneal injury. In the present study, we questioned whether AM also might be a source of TFF3 and if yes whether the secretion rate of TFF3 is changed by proinflammatory cytokines or by cryoconservation of AM. By means of RT-PCR, the mRNA expression of all three known TFF peptides could be detected in AM. Immunohistochemistry on paraffin-embedded sections localized TFF3 protein and also TFF2 in AM cells and Western blot analysis revealed TFF3 protein in AM. Stimulation experiments with proinflammatory cytokines and subsequent TFF3 ELISA measurements revealed that the secretion rate of fresh or cryoconserved AM was not significantly changed. The results indicate that TFF peptides are produced by AM. TFF3 may contribute to ocular surface wound healing after AM transplantation, but its production by AM is not further inducible by proinflammatory stimuli. Cryopreservation has no effect on the secretion rate of TFF3 supporting the use of cryopreserved AM for transplantation.

[Alterations of the anterior segment of the eye caused by exposure to UV radiation]. / UV-strahlenexpositionsbedingte Veränderungen am vorderen Augenabschnitt.

BACKGROUND: By identifying diseases of the anterior segment of the eye associated with exposure to UV light, recommendations for action can be derived. AIM: After reading this review, the reader should be familiar with UV light-associated diseases of the anterior segment of the eye. METHOD: Using a selective literature search, UV light-associated diseases of the anterior segment of the eye were identified and protective mechanisms are described. RESULTS: The UV light-associated lesions of the anterior segment of the eye include basal cell and squamous cell carcinomas, malignant melanoma of the eyelids and conjunctiva, pterygium, keratoconjunctivitis photoelectrica and climatic droplet keratopathy as well as cortical cataract. CONCLUSION: Eyeglasses for filtering UV light, sunglasses and special safety glasses, such as welding helmets and wearing headgear protect against UV light exposure to the anterior segment of the eye and the associated diseases.

Dry Eye Parameters and Lid Geometry in Adults Born Extremely, Very, and Moderately Preterm with and without ROP: Results from the Gutenberg Prematurity Eye Study.

Background/Aims: This study aimed to analyze the effects of perinatal history on tear film properties and lid geometry in adults born preterm. Methods: The Gutenberg Prematurity Eye Study (GPES) is a German prospective examination of adults born preterm and term aged 18 to 52 years with Keratograph® 5M and Schirmer test I. Main outcome measures were first non-invasive tear film break-up time (F-NITBUT), bulbar redness (BR), Schirmer test, and nasal palpebral angle measurement. The associations with gestational age (GA), birth weight (BW), and BW percentile, retinopathy of prematurity (ROP), ROP treatment, and other perinatal factors were evaluated using regression analyses. Results: 489 eyes of 255 preterm and 277 eyes of 139 full-term individuals (aged 28.6 +/− 8.8 years, 220 females) were included. Of these, 33 participants (56 eyes) had a history of spontaneously regressed ROP and 9 participants (16 eyes) had a history of ROP treatment. After adjustment for age and sex, lower F-NITBUT (<20 s) was associated with ROP treatment (OR = 4.42; p = 0.025). Lower GA correlated with increased bulbar redness (B = −0.02; p = 0.011) and increased length of wetting in the Schirmer test (B = −0.69; p = 0.003). Furthermore, low GA was associated with narrowing of the nasal palpebral angle (B = 0.22; p = 0.011) adjusted for age and sex, but not when considering ROP in the multivariable model. Conclusion: Our analyses indicate that perinatal history affects ocular surface properties, tear production and lid geometry in adults born term and preterm. This might indicate that affected persons have a predisposition to diseases of the corneal surface such as the dry eye disease.

Prevalence of corneal arcus and associated factors in a German population-Results from the Gutenberg Health Study.

PURPOSE: We aimed to determine the prevalence of corneal arcus and to identify associated factors in the general population of Germany. METHODS: The Gutenberg Health Study (GHS) is a population-based cohort study in Germany, which includes an ophthalmological assessment. Refraction, distance-corrected visual acuity, non-contact tonometry and anterior segment imaging were performed for the five-year follow-up examination. Anterior segment photographs were graded for the presence of corneal arcus. Prevalence estimates were computed, and multivariable logistic regression analysis was applied to determine associated factors for corneal arcus including sex, age, spherical equivalent, central corneal thickness, intraocular pressure (IOP), socio-economic status, smoking, BMI, systolic and diastolic arterial blood pressure, HbA1c, HDL-C, LDL-C, triglyceride, and lipid modifying agents. RESULTS: A total of 9,850 right and 9,745 left eyes of 9,858 subjects (59.2±10.8 years), 49.0% females were included in this cross-sectional analysis. 21.1% of men (95%-CI: 20.0%- 22.3%) had a corneal arcus in at least one eye, and 16.9% (95%-CI: 15.9%- 18.0%) of women. In multivariable analyses, the presence of corneal arcus was associated with male gender (OR = 0.54 for female, p<0.0001), higher age (OR = 2.54 per decade, p<0.0001), smoking (OR = 1.59, p<0.0001), hyperopia (OR = 1.05 per diopter, p<0.0001), thinner cornea (OR = 0.994 per µm, p<0.0001), higher IOP (OR = 1.02, p = 0.039), higher HDL-C-level (OR = 2.13, p<0.0001), higher LDL-C-level (OR = 1.21, p<0.0001), and intake of lipid modifying agents (OR = 1.26, p = 0.0001). Arcus was not associated with socio-economic status, BMI, arterial blood pressure, and HbA1c. CONCLUSIONS: Corneal arcus is a frequent alteration of the cornea in Germany and is associated with ocular parameters and systemic parameters of dyslipidemia.

Aggregated neutrophil extracellular traps occlude Meibomian glands during ocular surface inflammation.

PURPOSE: Obstructive Meibomian gland dysfunction (MGD) is one of the leading causes of evaporative dry eye disease. Meibomian glands at the eyelid secrete lipids that prevent evaporation of the aqueous tear film. The pathogenesis of obstructive MGD is incompletely understood to date. Herein, we aim to investigate the pathogenesis of obstructive MGD using murine and human samples with various forms of ocular surface inflammation. METHOD: The presence of Neutrophil extracellular Traps (NETs) was detected with immunofluorescence analysis of ocular surface discharge and biopsy samples from patients with blepharitis. Tear fluid from patients with MGD and blepharitis were evaluated for the presence of inflammatory mediators using bead based immunoassay. Murine model of allergic eye disease (AED) was performed to investigate the role of NETs in MG occlusion. RESULTS: we show that the ocular discharge from patients with blepharitis contains aggregated neutrophil extracellular traps (aggNETs). Furthermore, the ducts of human Meibomian glands affected by blepharitis were largely congested by aggNETs. Tear fluid from patients with MGD showed elevated neutrophil chemoattractants (C5a, IL6, IL8 and IL18). C5a and IL8 correlated with the degree of deficiency of tear fluid. In the murine model of allergic eye disease (AED), aggNETs accumulated in the MG leading to occlusion of their ducts and the retrograde pent-up of the fluid followed by acinar atrophy. Constraining aggNET formation by genetic or pharmacological inhibition of peptidyl arginine deiminase type 4 (PADI4) effectively reduced MG damage. CONCLUSION: We conclude that aggNETs occlude MG causing MGD after ocular surface inflammation.

Ocular Phenotype of Relaxin Gene Knockout (Rln-/-) Mice.

Purpose: To test if relaxin deficiency affects ocular structure and function we investigated expression of relaxin (Rln) and RXFP receptors (Rxfp1, Rxfp2), and compared ocular phenotypes in relaxin gene knockout (Rln-/- ) and wild type (Rln+/+ ) mice. Materials and Methods: Rln, Rxfp1 and Rxfp2 mRNA expression was detected in ocular tissues of Rln+/+ mice using RT-PCR. The eyes of 11 Rln-/- and 5 Rln+/+ male mice were investigated. Corneal and retinal thickness was assessed using optical coherence tomography. Intraocular pressure was measured using a rebound tonometer. Retinal, choroidal and sclera morphology and thickness were evaluated histologically. Eyes were collected and fixed for immunofluorescence staining or used for RNA extraction to evaluate mRNA expression using real-time PCR. Results: Rln mRNA was expressed only in the retina, whereas Rxfp1 transcripts were detected in the retina, cornea and sclera/choroid. Rxfp2 was only present in the cornea. None of these genes were expressed in the lacrimal gland, eyelid or lens. Intraocular pressure was higher and central cornea of Rln-/- mice was significantly thicker and had significantly larger endothelial cells and a lower endothelial cell density than Rln+/+ mice. Immunohistochemistry demonstrated no significant difference in AQP3 and AQP5 staining in the cornea or other regions between wildtype and Rln-/- mice. mRNA expression of Aqp4 was significantly higher in Rln-/- than in Rln+/+ corneas, whereas Col1a2, Mmp9, Timp1 and Timp2 were significantly decreased. Expression of Aqp1, Aqp4, Aqp5, Vim and Tjp1 was significantly decreased in Rln-/- compared to Rln+/+ uvea. No significant differences in these genes were detected in the retina. Retinal, choroidal and scleral thicknesses were not different and morphology appeared normal. Conclusion: The findings indicate that loss of Rln affects expression of several genes in the uvea and cornea and results in thicker corneas with altered endothelial cells. Many of the gene changes suggest alterations in extracellular matrix and fluid transport between cells.

Schirmer test results: are they associated with topical or systemic medication?

PURPOSE: To test whether Schirmer test (ST) results are associated with topical or systemic medication and to evaluate the distribution of tear fluid quantity in a 3-min and 5-min ST. METHODS: The Gutenberg Health Study is a population-based, prospective, observational cohort study in Germany. ST was assessed in a sub-cohort of 1,999 participants. ST was performed under topical anesthesia for 5 min (ST-5) or of 3 min (ST-3). Anthropometric factors, systemic diseases, use of systemic and eye medications were recorded. We used multivariable quantile regression analysis to assess the influence on ST measurements. RESULTS: The length of wetting of the Schirmer strips for ST-5 was 23.2 ±â€¯9.31 mm for right and 22.9 ±â€¯9.0 mm for left eyes. In ST-3, the measurements were 20.0 mm in right and 19.1 mm in left eyes. The clinical cut off of 10 mm for ST-5 corresponded with an 8 mm cut off for ST-3. There was an association of smaller ST-5 measures with male sex, higher age, socioeconomic status and season (all p < 0.001), but not with diabetes or smoking. The use of prostaglandin or beta-blocker eye drops or oral non-steroidal anti-inflammatory drugs, drugs for peptic ulcer and gastro-oesophageal reflux disease, thyroid hormones, progesterone and estrogen combination drugs, and hypnotics and sedatives showed an association with smaller ST-5. CONCLUSIONS: For the first time we describe the distribution of tear fluid quantity by ST in a very large cohort of the general population. Furthermore, we found associations of ST measures with topical and systemic medication.

Ophthalmological Findings in Mucopolysaccharidoses.

The mucopolysaccharidoses (MPS) are a heterogenous group of lysosomal storage disorders caused by the accumulation of glycosaminoglycans (GAGs). The accrual of these compounds results in phenotypically varied syndromes that produce multi-organ impairment with widespread systemic effects. The low incidence of MPS (approximately 1/25,000 live births) in conjunction with the high childhood mortality rate had limited the availability of research into certain clinical features, especially ocular manifestations. As the recent successes of hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) have greatly increased life expectancy in these patients, they have served as a focal point for the transition of research towards improvement of quality of life. Ophthalmological findings in MPS include corneal clouding, glaucoma, optic neuropathies, and retinopathies. While corneal clouding is the most common ocular feature of MPS (especially type I, IVA, and VI), its response to HSCT and ERT is minimal. This review discusses known eye issues in the MPS subtypes, diagnosis of these ocular diseases, current clinical and surgical management, noteworthy research progress, and ultimately presents a direction for future studies.

In vitro effects of benzalkonium chloride and prostaglandins on human meibomian gland epithelial cells.

PURPOSE: Benzalkonium chloride is the most widely used preservative in ophthalmic topical solutions. The aim of this study was to investigate the influence of BAC as a single substance or as a component of several commercially available ophthalmic solutions on meibomian gland epithelial cells in vitro. MATERIALS AND METHODS: An immortalized human meibomian gland epithelial cell line (HMGEC) was used and cells were cultured in the absence or presence of fetal bovine serum to assess cell morphology, cell proliferation, cell viability (MTS assay) and impedance sensing (ECIS) after stimulation with BAC. Further, the viability of HMGECs stimulated with BAC-containing and BAC-free bimatoprost, travoprost and latanoprost was evaluated using the MTS assay. Real-time PCR analysis for hyperkeratinization associated genes (cornulin, involucrin) was performed. RESULTS: In the absence of serum, the proliferation rate of HMGECs decreased starting with 0.1µg/ml BAC. At concentrations of 50µg/ml BAC and higher, cell viability was reduced after 10min exposure with a corresponding change in cell morphology. Toxicity of BAC-containing ophthalmic solutions was greater than that of BAC alone, whereas BAC-free alternative products did not significantly influence cell viability. Confluence, cell-cell contacts and serum-containing medium appeared to facilitate HMGECs survival. Expression rate of involucrin and cornulin declined after exposure to preserved bimatoprost and BAC. CONCLUSIONS: BAC showed cytotoxic effects on HMGECs starting with a concentration of 0.1µg/ml. The combination of BAC and prostaglandin-analogs might have a synergistic effect which results in higher toxicity than BAC alone. Unpreserved eye drops and eye drops preserved with Polyquaternium-1 are less damaging to HMGECs.

Effect of intermittent shear stress on corneal epithelial cells using an in vitro flow culture model.

PURPOSE: The aim of this study was to establish and to evaluate an in vitro model for culturing human telomerase-immortalized corneal epithelial (hTCEpi) cells under adjustable medium flow mimicking the movements of the tear film on the ocular surface. METHODS: Using an IBIDI pump system, cells were cultured under unidirectional, continuous or oscillating, discontinuous medium flow. Cell surface and cytoskeletal architecture were investigated by scanning electron microscopy and immunofluorescence. Gene expression of e-cadherin, occludin, tight junction protein (TJP), desmoplakin, desmocollin and mucins was investigated by real-time PCR. Protein expression of desmoplakin, TJP, occludin and e-cadherin was analyzed by western blot and localization was detected by immunofluorescence. Rose bengal staining was used to assess mucin (MUC) barrier integrity. MUC1, -4 and -16 proteins were localized by immunofluorescence. RESULTS: Medium flow-induced shear stress dramatically changed cellular morphology of hTCEpi. Cells subjected to discontinuous shear stress displayed the typical flattened, polygonal cell shape of the superficial layer of stratified squamous epithelia. Cell surfaces showed less bulging under shear stress and less extracellular gaps. The mRNA expression of E-cadherin, occludin and TJP were increased under oscillatory medium flow. Desmoplakin and occludin protein were upregulated under oscillatory shear stress. Stress fiber formation was not aligned to flow direction. MUC1, -4, and -16 protein were localized under all culture conditions, a regulation on mRNA expression was not detectable. Rose Bengal uptake was diminished under unidirectional conditions. CONCLUSION: Our findings suggest that shear stress as it occurs at the ocular surface during blinking exerts marked effects on corneal epithelial cells, such as changes in cellular morphology and expression of cell junctions. The described model may be useful for in vitro investigations of ocular surface epithelia as it represents a much more physiologic reproduction of the in vivo situation than the commonly applied static culture conditions.

Differentiation Patterns of Immortalized Human Meibomian Gland Epithelial Cells in Three-Dimensional Culture.

Purpose: To establish a simplified three-dimensional (3D) meibomian gland culture model using a meibomian gland epithelial cell (HMGEC) line that might be a useful tool to gain deeper insights into meibomian gland dysfunction. For this purpose, 3D differentiation patterns and growth characteristics of HMGECs were studied on various membranes/scaffolds as well as in hanging drops. Methods: Several types of inserts consisting of different materials (Millicell-HA, Millicell-PCF, ThinCert, and Alvetex) as well as hanging drop culture were analyzed. Culture conditions were optimized employing exposure to air (air-lift) and different cell culture media for a maximum of 28 days. To characterize cell differentiation in the developed 3D model, the expression pattern of cytokeratins was investigated by immunohistochemistry. Sudan III staining was performed for detection of lipid formation and transmission electron microscopy (TEM) was used for ultrastructural analysis. Results: Only Alvetex scaffolds and the hanging drop method revealed satisfactory results with regard to 3D culture. Continuous use of proliferation medium (serum-free keratinocyte medium containing epidermal growth factor and bovine pituitary extract) and air-lift were important steps for HMGEC differentiation in 3D culture. However, HMGECs only reached a differentiating state and never became mature or hypermature. When cultured in hanging drops, HMGECs showed serum-induced keratinization processes. Conclusions: HMGECs have the capability to differentiate in a long-term 3D culture, especially when adapted to an air-rich environment. However, even in the 3D format, HMGECs only reach a state of differentiating meibocytes.

Does endogenous serum oestrogen play a role in meibomian gland dysfunction in postmenopausal women with dry eye?

AIM: To explore the relationship between serum concentration of sex hormones and dry eye symptoms and signs in postmenopausal women. METHODS: A cross-sectional analysis was undertaken. Subjects were 46 postmenopausal women with dry eye (mean age 64.4±5.2 years, 13.7±6.4 years since menopause; not undergoing hormone replacement therapy). Ocular symptoms (Ocular Surface Disease Index (OSDI) and Ocular Comfort Index (OCI)), tear function (tear osmolarity, non-invasive tear break-up time, tear secretion), corneal and conjunctival staining, and meibomian gland (MG) appearance, were recorded. Venous blood was collected and serum concentrations of 17ß-oestradiol (E2), 3-α-androstanediol-glucuronide (3α-diol-G), and dehydroepiandrosterone sulfate (DHEA-S) were determined using ELISA. Multiple linear regression analysis was used to examine predictors of dry eye symptoms and signs. RESULTS: Mean serum concentration of E2, 3α-diol-G and DHEA-S was 9.02±13.40 pg/mL, 1.59±1.02 ng/mL and 0.74±0.53 µg/mL, respectively. Ocular symptoms were elevated (mean scores 27.0±18.1 (OSDI) and 40.3±8.4 (OCI)) but signs were within normal ranges. Higher serum E2 concentration along with capped glands, lid telangiectasia and older age was a significant predictor of worse MG secretion quality (p<0.001, R2adj=0.75). Serum hormones were not significant predictors of ocular symptoms in multivariate analysis (p>0.05). CONCLUSION: Serum oestrogen appears to be a key factor in MG signs. Although serum hormone levels did not contribute significantly to dry eye symptoms in this study, it is possible that oestrogen plays a role through its effect on meibum secretion. These findings suggest that MG dysfunction underpins dry eye symptoms in non-Sjögren's dry eye in postmenopausal women. TRIAL REGISTRATION NUMBER: ACTRN12612000281897.

The effects of transdermal testosterone and oestrogen therapy on dry eye in postmenopausal women: a randomised, placebo-controlled, pilot study.

AIMS: Sex hormones could provide a future treatment avenue for dry eye post menopause. However, there are few well-controlled studies. This study investigates the impact of testosterone and oestrogen on dry eye symptoms and signs in postmenopausal women. METHODS: A randomised double-blind placebo-controlled pilot study was conducted involving 40 women with dry eye (age 63.9±5.1 years, 13.2±6.3 years post menopause). Ten women were assigned to each of four treatment groups: transdermal testosterone, oestradiol, testosterone/oestradiol combination and placebo. Assessment at baseline and after 8 weeks: ocular symptoms, tear osmolarity, tear stability, tear secretion, meibomian gland assessment, corneal and conjunctival sensitivity, serum concentrations of 17ß-oestradiol, 3-α-androstanediol-glucuronide and dehydroepiandrosterone sulfate. Differences from placebo were examined using one-way analysis of variance and Dunnett's t-test. Within-group analyses included paired t-tests and Spearman correlation. RESULTS: Dryness intensity after 8 weeks was significantly worse in the oestrogen group compared with placebo (p=0.04). No significant changes in other symptoms, tear function, meibomian gland function, lid morphology, corneal or conjunctival sensitivity were observed in any of the groups when compared with the change in placebo after 8 weeks. Within-group analyses showed increased tear secretion in the testosterone/oestradiol combination group (p=0.03) and a strong association between increased serum androgen and improved tear stability in the testosterone group (ρ=0.83,p=0.01). CONCLUSIONS: Oestrogen supplementation may worsen ocular symptoms in postmenopausal women with dry eye, whereas no impact of testosterone therapy on symptoms was apparent. The positive effects of oestrogen and testosterone on tear function require confirmation in a larger study, with sample size calculated from the data generated herein. Placebo control is essential in studies of dry eye therapies. TRIAL REGISTRATION NUMBER: ACTRN12612000281897.