Identification of the regulatory circuit governing corneal epithelial fate determination and disease.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.

A Cross-sectional Analysis of 556 Eyes Entering the Homburg Aniridia Centre.

PURPOSE: Congenital aniridia is a severe malformation of almost all eye segments. In addition, endocrinological, metabolic, and central nervous systems diseases may be present. In order to develop better treatment options for this rare disease, an aniridia center must be established. The purpose of this work is to summarize ophthalmic findings of aniridia subjects examined at the Department of Ophthalmology, Saarland University Medical Center in Homburg. METHODS: Our retrospective single-center study included patients who underwent a comprehensive ophthalmic examination through the head of the KiOLoN ("Kinderophthalmologie", Orthoptics, Low Vision and Neuroophthalmology) Unit of the department between June 2003 and January 2022. Data at the first examination time point have been included. RESULTS: Of 286 subjects, 556 eyes of (20.1 ± 20.1 years; 45.5% males) were included. There was nystagmus in 518 (93.7%) eyes, and strabismus in 327 (58.8%) eyes. There were 436 (78.4%) eyes with age-appropriate axial length, 104 (18.7%) eyes with microphthalmos, and 13 (2.3%) eyes with buphthalmos. There was iris malformation with atypical coloboma in 34 eyes (6.1%), more than 6 clock hours of iris remnants in 61 eyes (10.9%), less than 6 clock hours of iris remnants in 96 eyes (17.2%), and complete aniridia in 320 (57.5%) eyes. The patients were graded according to the following aniridia-associated keratopathy (AAK) stages: Stage 0 (96 eyes [17.2%], no keratopathy), Stage 1 (178 eyes [32.0%]), Stage 2 (107 eyes [19.2%]), Stage 3 (67 eyes [12.0%]), Stage 4 (62 eyes [11.1%]), Stage 5 (45 eyes [8.0%]). There was secondary glaucoma in 307 (55.5%), macular hypoplasia in 395 (71.4%), and congenital optic nerve head pathology in 223 (40.3%) eyes. The iris malformation type was significantly positively correlated with AAK stage, lens properties, presence of glaucoma, congenital macular, and optic nerve head properties (p < 0.001 for all), while complete aniridia showed the most complications. CONCLUSIONS: At the Homburg Aniridia Center, the most common ophthalmic signs in congenital aniridia were AAK, iris malformation, cataract, and macular hypoplasia. The iris malformation type may indicate future expression of AAK, cataract, and glaucoma development and it is correlated with a congenital optic nerve head and macular pathology. Our registry will support further detailed longitudinal analysis of ophthalmic and systemic diseases of aniridia subjects during long-term follow-up.

Assessment of Rose Bengal Photodynamic Therapy on Viability and Proliferation of Human Keratolimbal Epithelial and Stromal Cells In Vitro.

PURPOSE: To investigate the effect of Rose Bengal photodynamic therapy (RB-PDT) on viability and proliferation of human limbal epithelial stem cells (T-LSCs), human corneal epithelial cells (HCE-T), human limbal fibroblasts (LFCs), and human normal and keratoconus fibroblasts (HCFs and KC-HCFs) in vitro. METHODS: T-LSCs and HCE-T cell lines were used in this research. LFCs were isolated from healthy donor corneal limbi (n = 5), HCFs from healthy human donor corneas (n = 5), and KC-HCFs from penetrating keratoplasties of keratoconus patients (n = 5). After cell culture, RB-PDT was performed using 0.001% RB concentration and 565 nm wavelength illumination with 0.14 to 0.7 J/cm2 fluence. The XTT and the BrdU assays were used to assess cell viability and proliferation 24 h after RB-PDT. RESULTS: RB or illumination alone did not change cell viability or proliferation in any of the cell types (p ≥ 0.1). However, following RB-PDT, viability decreased significantly from 0.17 J/cm2 fluence in HCFs (p < 0.001) and KC-HCFs (p < 0.0001), and from 0.35 J/cm2 fluence in T-LSCs (p < 0.001), HCE-T (p < 0.05), and LFCs ((p < 0.0001). Cell proliferation decreased significantly from 0.14 J/cm2 fluence in T-LSCs (p < 0.0001), HCE-T (p < 0.05), and KC-HCFs (p < 0.001) and from 0.17 J/cm2 fluence in HCFs (p < 0.05). Regarding LFCs proliferation, no values could be determined by the BrdU assay. CONCLUSIONS: Though RB-PDT seems to be a safe and effective treatment method in vivo, its dose-dependent phototoxicity on corneal epithelial and stromal cells has to be respected. The data and experimental parameters applied in this study may provide a reliable reference for future investigations.

Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro - A Stepwise Linear Regression Algorithm.

PURPOSE: To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm. METHODS: Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO2 atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm. RESULTS: The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007). CONCLUSIONS: The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro.

The Effect of Glaucoma Treatment on Aniridia-Associated Keratopathy (AAK) - A Report from the Homburg Register for Congenital Aniridia.

BACKGROUND: Congenital aniridia is a severe malformation of almost all eye segments. Aniridia-associated keratopathy (AAK) and secondary glaucoma, which occur in more than 50% of affected individuals, are typically progressive and pose a high risk of blindness for patients with congenital aniridia. Our aim was to investigate the effect of glaucoma treatment on AAK in patients of the Homburg Aniridia Center. METHODS: Our retrospective monocentric study included patients who underwent a comprehensive ophthalmological examination at the Homburg Aniridia Center between June 2003 and January 2022. RESULTS: There were 556 eyes of 286 subjects (20.1 ± 20.1 years; 45.5% males) included. In 307 (55.2%) eyes of 163 subjects (27.5 ± 16.3 years; 43.1% males), glaucoma was present at the time of examination. The mean intraocular pressure in the glaucoma group was 19.0 mmHg (± 8.0), while in the non-glaucoma group, it was 14.1 mmHg (± 3.6) (p < 0.001). In the glaucoma group, 68 patients used antiglaucomatous topical monotherapy, 51 patients used 2 agents, 41 patients used 3 agents, 7 patients used quadruple therapy, and 140 did not use topical therapy (e.g., after pressure-lowering surgery, pain-free end-stage glaucoma, or incompliance). Patients were classified according to the following stages of AAK: Stage 0 (96 eyes [17.2%], no keratopathy), Stage 1 (178 eyes [32.0%]), Stage 2 (107 eyes [19.2%]), Stage 3 (67 eyes [12.0%]), Stage 4 (62 eyes [11.1%]), Stage 5 (45 eyes [8.0%]). The mean stage of AAK was 1.4 (1.2 - 1.5) in the group without eye drops, 1.9 (1.5 - 2.2) in the group with monotherapy, 1.8 (1.5 - 2.1) in the group with 2 drugs, 1.9 (1.5 - 2.2) in the group with 3 drugs, 3.4 (2.3 - 4.6) in the group with 4 drugs, and 3.3 (3.1 - 3.6) after antiglaucomatous surgery. The stage of AAK was significantly positively correlated with the number of pressure-lowering eye drops (p < 0.05) and prior pressure-lowering surgery (p < 0.05). Prostaglandin analogues were not correlated with a higher AAK stage compared to the other drug groups. CONCLUSIONS: At the Homburg Aniridia Center, patients using topical antiglaucomatous quadruple therapy or who had previously undergone antiglaucomatous surgery had by far the highest AAK stage. The different drug groups had no influence on the AAK stage.

Decreased FABP5 and DSG1 protein expression following PAX6 knockdown of differentiated human limbal epithelial cells.

PAX6 haploinsufficiency related aniridia is characterized by disorder of limbal epithelial cells (LECs) and aniridia related keratopathy. In the limbal epithelial cells of aniridia patients, deregulated retinoic acid (RA) signaling components were identified. We aimed to visualize differentiation marker and RA signaling component expression in LECs, combining a differentiation triggering growth condition with a small interfering RNA (siRNA) based aniridia cell model (PAX6 knock down). Primary LECs were isolated from corneoscleral rims of healthy donors and cultured in serum free low Ca2+ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca2+. In addition, LECs were treated with siRNA against PAX6. DSG1, PAX6, KRT12, KRT 3, ADH7, RDH10, ALDH1A1, ALDH3A1, STRA6, CYP1B1, RBP1, CRABP2, FABP5, PPARG, VEGFA and ELOVL7 expression was determined using qPCR and western blot. DSG1, FABP5, ADH7, ALDH1A1, RBP1, CRABP2 and PAX6 mRNA and FABP5 protein expression increased (p ≤ 0.03), PPARG, CYP1B1 mRNA expression decreased (p ≤ 0.0003) and DSG1 protein expression was only visible after Ca2+ supplementation. After PAX6 knock down and Ca2+ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca2+ supplementation alone. Using our cell model, with Ca2+ supplementation and PAX6 knockdown with siRNA treatment against PAX6, we provide evidence that haploinsufficiency of the master regulatory gene PAX6 contributes to differentiation defect in the corneal epithelium through alterations of RA signalling. Upon PAX6 knockdown, DSG1 differentiation marker and FABP5 RA signaling component mRNA expression decreases. A similar effect becomes apparent at protein level though differentiation triggering Ca2+ supplementation in the siRNA-based aniridia cell model. Expression data from this cell model and from our siRNA aniridia cell model strongly indicate that FABP5 expression is PAX6 dependent. These new findings may lead to a better understanding of differentiation processes in LECs and are able to explain the insufficient cell function in AAK.

Endothelial Cell Density and Central Corneal Thickness following Penetrating Keratoplasty of Acanthamoeba Keratitis Patients - A Retrospective Cross-Sectional Observational Study.

PURPOSE: To analyze endothelial cell density (ECD) and central corneal thickness (CCT) following penetrating keratoplasty (PKP) in Acanthamoeba keratitis (AK) patients. PATIENTS AND METHODS: In this retrospective, clinical, single-center, cross-sectional, observational study, patients were enrolled who underwent PKP at the Department of Ophthalmology of Saarland University Medical Center, Homburg/Saar, Germany between May 2008 and December 2016 with the diagnosis of AK. In all, 33 eyes of 33 patients (14 males, 42%) were enrolled; their mean age at the time of surgery was 39.5 ± 14.3 years. Postoperatively, AK patients received topical polyhexamethylene biguanide, propamidine isethionate, neomycin sulphate/gramicidin/polymixin B sulfate, and prednisolone acetate eye drops (5 ×/day each), and the topical treatment was tapered sequentially with 1 drop every 6 weeks over 6 months. CCT was recorded using Pentacam HR Scheimpflug tomography and ECD with the EM-3000 specular microscope before surgery and 3 and 6 months after surgery as well as after the first and second (complete) suture removal. RESULTS: ECD tended to decrease significantly from the time point before surgery (2232 ± 296 cells/mm2) to the time point 3 months after surgery (1914 ± 164 cells/mm2; p = 0.080) and to the time point after the first suture removal (1886 ± 557 cells/mm2; p = 0.066) and decrease significantly to the time point after the second suture removal (1650 ± 446 cells/mm2; p = 0.028). CCT did not change significantly over the analyzed time period (p ≥ 0.475). CONCLUSION: In AK, endothelial cell loss does not seem to be accelerated following PKP, despite the postoperative use of diamidine and biguanide. A subsequent prospective comparative study should confirm our retrospective longitudinal analysis.

Different mRNA expression patterns in keratoglobus and pellucid marginal degeneration keratocytes.

PURPOSE: Alike keratoconus (KC), keratoglobus (KG) and pellucid marginal degeneration (PMD) belong to ectatic corneal diseases. While there are numerous studies on keratoconus pathophysiology, there is no exact knowledge on genetic and pathophysiological background of KG and PMD, so far. It is not yet clarified, whether KG and PMD are independent clinical entities or represent different stages of the same disease. Our purpose was to investigate key parameters concerning collagen synthesis, intracellular LOX expression and inflammation in corneal stromal cells of KG and PMD subjects, in vitro. METHODS: Normal human keratocytes of corneas from the LIONS Cornea Bank Saar-Lor-Lux, Trier/Westpfalz and human keratocytes of KG and PMD patients were isolated and cultured as keratocytes. To examine Collagen I and V (Col I, Col V), heat shock protein 47 (Hsp47), Lysyl Oxidase (LOX), nuclear factor kappa B (NF-κB) mRNA and protein expression in all cell types, quantitative PCR and Western blot analysis has been performed. RESULTS: Col5A1 mRNA expression was significantly lower in KG and PMD keratocytes and LOX mRNA expression was significantly higher in KG-keratocytes, compared to controls. Col1A1, Hsp47 and NF-κB mRNA expression and the analyzed protein expressions did not differ from controls, in KG or PMD. CONCLUSIONS: Col5A1 mRNA expression is decreased in KG and PMD and LOX mRNA expression is increased in KG. Therefore, the pathophysiology of KG and PMD differs from KC and these seem to be from KC independent entities. The explanation of the peripheral corneal thinning in KG and PMD must be investigated in further studies.

Hypoxic stress increases NF-κB and iNOS mRNA expression in normal, but not in keratoconus corneal fibroblasts.

BACKGROUND: Keratoconus (KC) is associated with oxidative stress and hypoxia and as several times discussed, potentially with inflammatory components. Inflammation, hypoxia, and oxidative stress may result in metabolic dysfunction and are directly linked to each other. In the current study, we investigate the effect of hypoxia through NF-κB signaling pathways on iNOS, hypoxia-induced factors (HIF), ROS, and proliferation of normal and KC human corneal fibroblasts (HCFs), in vitro. METHODS: Primary human KC-HCFs and normal HCFs were isolated and cultured in DMEM/Ham's F12 medium supplemented with 5% fetal calf serum. Hypoxic conditions were generated and quantitative PCR and Western blot analysis were performed to examine NF-κB, iNOS, HIF, and PHD2 expression in KC and normal HCFs. ROS level was analyzed using flow cytometry and proliferation by BrdU-ELISA. RESULTS: Hypoxia increased NF-κB mRNA and protein expression in normal HCFs, but in KC-HCFs NF-κB mRNA and protein expression remained unchanged. Hypoxic conditions upregulated iNOS mRNA expression of normal HCFs, but iNOS mRNA expression of KC-HCFs and iNOS protein expression of both HCF types remained unchanged. Hypoxia downregulated HIF-1α and HIF-2α mRNA expression in normal and KC-HCFs. PHD2 mRNA expression is upregulated under hypoxia in KC-HCFs, but not in normal HCFs. PHD2 protein expression was upregulated by hypoxia in both HCF types. Total ROS concentration is downregulated in normal and KC-HCFs under hypoxic conditions. Proliferation rate of KC-HCFs was upregulated through hypoxia, but did not change in normal HCFs. CONCLUSIONS: Hypoxia increases NF-κB and iNOS mRNA expression in normal HCFs, but there does not seem to be enough capacity in KC-HCFs to increase NF-κB and iNOS mRNA expression under hypoxia, maybe due to the preexisting oxidative stress. HIF and PHD2 do not show altered iNOS regulation under hypoxic conditions in KC-HCFs, and therefore do not seem to play a role in keratoconus pathogenesis. An increased proliferation of cells may refer to compensatory mechanisms under hypoxia in KC. Understanding the mechanism of the altered regulation of NF-κB and iNOS in KC-HCFs will provide better insight into the potential inflammatory component of the KC pathogenesis.

NF-κB, iNOS, IL-6, and collagen 1 and 5 expression in healthy and keratoconus corneal fibroblasts after 0.1% riboflavin UV-A illumination.

PURPOSE: To analyze the effect of riboflavin UV-A illumination on mRNA and protein expression of healthy (HCFs) and keratoconus human corneal fibroblasts (KC-HCFs), concerning the inflammatory markers NF-κB, iNOS, IL-6, and collagen 1 and 5 (Col 1/Col 5). METHODS: Keratocytes were isolated from healthy (n = 3) and keratoconus (KC) corneas (n = 3) and were cultivated in basal medium with 5% fetal calf serum, which resulted in their transformation into human corneal fibroblasts (HCFs/KC-HCFs). Cells underwent 0.1% riboflavin UV-A illumination for 250 s (CXL). NF-κB, iNOS, IL-6, Col 1, and Col 5 expression was investigated by qPCR and Western blot analysis. IL-6 concentration of the cell culture supernatant and cell lysate was determined by ELISA. RESULTS: In untreated KC-HCFs, NF-κB (p = 0.0002), iNOS (p = 0.0019), Col 1 (p = 0.0286), and Col 5 (p = 0.0054) mRNA expression was higher and IL-6 expression was lower (p = 0.0057), than in healthy controls. In HCFs, CXL led to an increased NF-κB (p = 0.0286) and IL-6 (p = 0.0057) mRNA expression. The IL-6 concentration in the cell culture supernatant was increased in HCFs (p = 0.0485) and KC-HCFs (p = 0.0485) after CXL. CXL increased intracellular IL-6 concentration only in KC-HCFs (p = 0.0357). In the HCF group (p = 0.0286), an increased Col 1 mRNA expression after CXL could be observed. CONCLUSION: Our study confirmed altered gene expression in untreated KC-HCFs compared to untreated HCFs. Riboflavin UV-A illumination affected gene expression only in HCFs. Increased IL-6 concentration in the cell culture supernatant and cell lysate indicate a secondary inflammatory response of HCFs and KC-HCFs to riboflavin UV-A illumination.

[Increased NF-κB and iNOS Expression in Keratoconus Keratocytes - Hints for an Inflammatory Component?] / Erhöhte NF-κB- und iNOS-Expression in Keratozyten von Keratokonuspatienten ­ Hinweise auf eine entzündliche Komponente?

PURPOSE: In recent years, there has been increasing evidence of an inflammatory component in keratoconus. A key gene in inflammatory processes is the nuclear factor kappa B (NF-κB). NF-κB is a transcription factor for the enzyme nitric oxide synthase (NOS), which is involved with the competing enzyme arginase (Arg) in inflammatory processes. The aim of this study was to analyze the isotypes of NOS and arginase, the expression of NF-κB, NOS and arginase, and the regulatory mechanism of NOS and arginase in keratocytes of keratoconus patients using the inhibitor 1400W in vitro. METHODS: Human keratocytes were isolated from surgically removed corneas of 8 KC patients and 8 normal human corneal buttons and were cultured to confluence, in vitro. Quantitative PCR and Western blot analysis were performed to examine NF-κB, NOS and arginase expression in keratocytes. Nitrite and urea concentrations in the supernatant of the cells were analyzed using 0 - 40 µM 1400W iNOS inhibitor concentrations. RESULTS: Only the isotypes iNOS and Arg-II were detected in the keratocytes. The mRNA expression of NF-κB and iNOS were higher in KC keratocytes than in normal cells (p = 0.0135 and p = 0.0001), whereas no differences were measurable in Arg-II expression. In the WB, a higher band intensity was measurable in NF-κB (p = 0.0012), and in iNOS, no differences in band intensity could be detected. In the supernatant of the KC keratocytes, lower concentrations of nitrite and urea were measured after the addition of the inhibitor 1400W (p ≤ 0.014), but not in normal cells (p ≥ 0.178). CONCLUSION: Due to the increased expression of NF-κB and iNOS, an inflammatory component in keratoconus must be assumed. The different regulation of the KC keratocytes by the iNOS inhibitor 1400W suggests an altered metabolic activity which can be caused by inflammatory processes.

Impact of Pre- and Intraoperative Factors on Endothelial Cell Density in the Early and Late Stage after Penetrating Keratoplasty. / Prä- und intraoperative Einflussgrößen auf die Endothelzellentwicklung in der Früh- und Spätphase nach perforierender Keratoplastik.

AIM: This retrospective investigated the impact of donor age, recipient age, donor endothelial cell density, vis-à-tergo, and additional intraoperative lens exchange (triple-procedure) on overall early and late phase postoperative endothelial cell density (ECD) following penetrating keratoplasty (PKP) in various diagnosis groups. PATIENTS AND METHODS: In 590 cases with diagnosed keratoconus (KC), Fuchs dystrophy (FD) and herpes simplex virus infection (HSV) who underwent PKP or triple surgery, the ECD in cells/mm2 was analysed, both preoperatively, with all-sutures-in (early postoperative stage), and after last suture removal. The factors were tested by Mann-Whitney U-test, correlation analysis and linear regression analysis. OUTCOME: Correlation analysis demonstrated a weak negative correlation between the patient's ECD and donor age (early postoperative stage: r = - 0.25, p < 0.001; after last suture removal: r = - 0.16; p = 0.003). Regression analysis revealed that donor age did not impact postoperative patient ECD. There was a weak negative correlation between postoperative ECD and recipient age (early postoperative stage: r = - 0.31, p < 0.001; after last suture removal: r = - 0.34, p < 0.001). Regression analysis confirmed the negative impact of recipient age on patient ECD (early postoperative stage: ß = - 13.2, p = 0.001; after last suture removal: ß = - 4.6, p < 0.001). Correlation analysis determined a weak positive correlation between postoperative ECD and donor endothelial cell density (early postoperative stage: r = 0.37, p < 0.001; after last suture removal: r = 0.32, p < 0.001). Regression analysis also determined that donor endothelial cell density had a positive impact on postoperative ECD following last suture removal (ß = 0.4, p < 0.001). Vis-à-tergo and additional lens exchange (triple procedure) had no significant effect on postoperative ECD (p > 0.05). This was also confirmed by the results of the regression analysis after last suture removal. CONCLUSION: Recipient age and donor endothelial cell density have a significant impact on postoperative ECD following PKP. Not all of the statistical tests proved donor age to be a significant influencing factor. Vis-à-tergo and additional lens exchange (triple procedure) had no significant effect on postoperative ECD following PKP.

Impact of COVID-19 Pandemic on Emergency Inpatient Volume at a Tertiary Eye Care Center in Germany with Corneal Main Specialization.

BACKGROUND: To estimate the impact of the COVID-19 pandemic on emergency inpatient volume in a tertiary eye care center in Germany with corneal main subspecialization. MATERIAL AND METHODS: A retrospective review of ocular emergency patients who attended the inpatient unit of the Department of Ophthalmology of Saarland University, Homburg/Saar, Germany during the COVID-19 pandemic, between 1 March and 30 April 2020, in comparison to the same time period in 2019. For each subject, clinical history and surgical reports were reviewed. After 24 March 2020, PCR examinations for SARS-CoV-2 were performed from throat swab specimens in all patients using real-time RT-PCR. RESULTS: Totally, 135 patients were admitted in 2019 and 115 patients in 2020 as emergency cases. The patient age at the time of admission did not differ significantly between the two time periods (63.6 ± 17.9 years vs. 62.5 ± 19.6 years) (p = 0.792), but the average length of hospital stays increased significantly for 2020 (4.0 ± 3.6 vs. 4.4 ± 2.7 days, p = 0.043). The percentage of admissions due to acute corneal hydrops (0% vs. 3.5%) increased significantly from 2019 to 2020 (χ2 = 4.772, p = 0.028), however, there was not a significant difference between the two years for any other diagnosis (χ2 ≤ 3.564, p ≥ 0.059). From 2019 to 2020, the percentage of acute intravitreal anti-VEGF injections decreased significantly (7.9% vs. 1.3%, χ2 = 3.985, p = 0.045), but the proportion of other emergency surgeries did not differ between the two years (χ2 ≤ 3.617, p ≥ 0.057). COVID-19 PCR examination was performed in 66 (57.4%) cases in 2020 and all samples (100%) were negative. CONCLUSIONS: The COVID pandemic did not change emergency inpatient volume in our department, but duration of hospital stay was extended on average by 8 hours, mainly due to additional COVID-19-PCR examinations. The proportion of the most frequently performed surgeries did not change remarkably between 2019 and 2020, but with the introduction of Muraine's sutures in 2019, the percentage of admissions with acute corneal hydrops (with or without subsequent surgery) increased for 2020. No urgent surgery had to be postponed due to the COVID-19 pandemic at our department; all operations were performed successfully.

HCE-T cell line lacks cornea-specific differentiation markers compared to primary limbal epithelial cells and differentiated corneal epithelium.

PURPOSE: Human corneal epithelial cell-transformed (HCE-T) cell line is used as a widely accepted barrier model for pharmacological investigations in the context of eye application. The differentiation of (limbal) corneal epithelial into mature corneal epithelium coincides with the expression of established differentiation markers. If these differentiation mechanisms are disturbed, it will lead to ocular surface disease. In this study, we want to compare the expression of differentiation markers in the HCE-T cell line to differentiated primary epithelial cells (pCECs) and primary limbal epithelial cell (LEC) culture. This is necessary in order to decide whether HCE-T cells could be a tool to study the differentiation process and its regulatory networks in corneal epithelium. METHODS: Primary limbal epithelial cells (LECs) for cell culture and primary corneal epithelial cells (pCECs) as differentiated tissue samples were obtained from the limbus or central cornea region of corneal donors. HCE-T cell line was purchased from RIKEN Institute RCB-2280.Expression levels of conjunctival- and corneal-specific keratin and adhesion markers (KRT3, KRT12, KRT13, KRT19, DSG1), stem cell and differentiation markers (PAX6, ABCG2, ADH7, TP63, ALDH1A1), and additional (unvalidated) putative differentiation and stem cell markers (CTSV, SPINK7, DKK1) were analyzed with qPCR. Additionally, KRT3, KRT12, DSG1, and PAX6 protein levels were analyzed with Western blot. RESULTS: KRT3, KRT12, DSG1, PAX6, ADH7, and ALDH1A1 mRNA expressions were higher in LECs and magnitudes higher in pCECs compared to HCE-T cells. KRT3, KRT12, PAX6, ALDH1A1, ADH7, TP63, and CTSV mRNAs have shown increasing mRNA expression from HCE-T < HCE-T cultured in keratinocyte serum-free medium (KSFM) < LEC < to pCEC.KRT3 and KRT12 protein expressions were only slightly increased in LEC compared to HCE-T samples, and the strongest signals were seen in pCEC samples. DSG1 protein expression was only detected in pCECs. PAX6 protein expression was hardly detected in HCE-T cells, and no difference could be seen between LECs and pCECs. CONCLUSIONS: The HCE-T cell line is even less differentiated than LECs regarding the investigated markers and therefore might also lack the ability to express differentiation markers at protein level. Hence, this cell line is not suitable to study corneal differentiation processes. Primary LECs in the way cultured here are not an ideal system compared to differentiated epithelium in organ culture but should be preferred to HCE-T cells if corneal differentiation markers are investigated. Other cell models or differentiation protocols should be developed in the future to gain new tools for research on ocular surface diseases.

Expression of retinoic acid signaling components ADH7 and ALDH1A1 is reduced in aniridia limbal epithelial cells and a siRNA primary cell based aniridia model.

PAX6-related Aniridia is a sight-threatening disease involving progression of secondary glaucoma and aniridia related keratopathy (ARK). Change or loss of limbal epithelial progenitors causes epithelial surface defects. We analyzed the effect of PAX6 on mRNA expression changes with a two-step approach, as follows. First, we sequenced mRNA from limbal epithelial cells isolated from controls and aniridia patients. Second, we confirmed the bioinformatics and literature-based result list for a small interfering RNA (siRNA)-based primary aniridia cell model (PAX6 knockdown). With this approach, we expected that the genes directly influenced by PAX6 would be distinguishable from those affected secondarily by the ARK disease state. Therefore, epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in keratinocyte serum-free medium. Normal control cells were obtained from the limbus region of corneal donors. For the siRNA-based aniridia cell model, cells were transfected with Lipofectamine and 5 nM siRNA against PAX6 or control treatment. All cells were lysed to yield DNA, RNA, and protein. Reduction of PAX6 protein was assessed by western blot. Aniridia and control Poly-A-enriched RNA libraries were subjected to next-generation sequencing. The differential analysis was a combination of quantification with RSEM and differential tests with edgeR. Gene lists were filtered by comparison to NCBI GEO datasets, annotated with DAVID, and manually annotated using a literature search. Based on the resulting filtered gene list, qPCR primers were purchased, and candidate genes (TP63, ABCG2, ADH7, ALDH1A1, PITX1, DKK1, DSG1, KRT12, KRT3, KRT13, SPINK6, SPINK7, CTSV, SERPINB1) were verified by qPCR on the siRNA-based aniridia cell model. We identified genes that might be regulated by PAX6 and showed that SPINK7 mRNA, which codes for a protease inhibitor, is downregulated in patients as well as in our primary aniridia cell model. ALDH1A1 and AHD7 mRNA levels were reduced in limbal epithelial cells of aniridia patients, and both transcripts were downregulated by PAX6 knockdown in our cell model. This siRNA-based aniridia cell model is a valuable tool for confirming identified PAX6-affected genes that might promote ARK pathogenesis. The model recapitulated expression changes for SPINK7, ADH7, and ALDH1A1 that were also observed in patient samples. These results provide evidence that PAX6 might drive corneal epithelial differentiation by direct or indirect control of retinoic acid signaling processes through ADH7 and ALDH1A1.

Comparison of in vitro assays to study the effectiveness of antiparasitics against Acanthamoeba castellani trophozoites and cysts.

We aimed to compare LDH release assay, trypan blue and fluorescent stainings, and non-nutrient Escherichia coli plate assay in determining treatment efficacy of antiamoebic agents against Acanthamoeba castellanii trophozoites/cysts, in vitro. 1BU trophozoites/cysts were challenged with 0.02% polyhexamethylene biguanid (PHMB), 0.1% propamidine isethionate (PD), and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH release and trypan blue assays, by Hoechst 33343, calcein-AM, and ethidium homodimer-1 fluorescent dyes, and by a non-nutrient agar E. coli plate assay. All three antiamoebic agents induced a significant LDH release from trophozoites, compared to controls (p < 0.0001). Fluorescent-dye staining in untreated 1BU trophozoites/cysts was negligible, but using antiamoebic agents, there was 59.3%-100% trypan blue, 100% Hoechst 33342, 0%-75.3% calcein-AM, and 100% ethidium homodimer-1 positivity. On E. coli plates, in controls and MF-treated 1BU trophozoites/cysts, new trophozoites appeared within 24 h, encystment occurred after 5 weeks. In PHMB- and PD-treated 1BU throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 h, which failed to form new cysts within 5 weeks. None of the enzymatic- and dye-based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non-nutrient agar E. coli plate assay, suggesting that the culture-based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.

Human aniridia limbal epithelial cells lack expression of keratins K3 and K12.

Aniridia is a rare disease of the eye that affects the iris, lens and the cornea. In about 90% of the cases, patients showed a loss of PAX6 function. Patients with aniridia often develop aniridia-related keratopathy (ARK), due to limbal stem cell insufficiency. The aim of this study was to determine the differentiation status of limbal epithelial cells (LECs) in patients with ARK. Epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium supplemented with EGF and BPE. Normal cells were obtained from limbus region of cadaveric control patients. Cells were analyzed with RT-PCR, qPCR and Western blot to evaluate expression of the developmental transcription factor, PAX6, potential stem cell markers, ΔNp63α and ABCG2, and corneal differentiation markers, keratin 12 (K12) and K3. Conjunctival differentiation markers, keratin 13 (K13) and K19 were also investigated. Cells were immunostained to evaluate K3, PAX6, and p63α protein expression. Protein coding sequence of PAX6 from patient LEC-cDNA was cloned and sequenced. RT-PCR showed that K3 and K12 transcripts were absent from patient cells, but present in healthy control preparations. Transcription levels of PAX6, ABCG2, and p63α of aniridia patients show no differences compared to normal control cells. Western blot showed reduced PAX6, protein levels in aniridia-LECs compared to control-LECs. Immunostaining also showed reduced PAX6 and K3 expression in aniridia-LECs compared to control-LECs. One aniridia patient showed a loss of stop codon in half of the cloned transcripts. In the second aniridia patient mRNA degradation through nonsense mediated decay seems to be very likely since we could not identify the mutation c.174C > T (Refseq. NM_000280), or misspliced transcripts in cDNA. We identified decreased PAX6 protein levels in aniridia patients in addition to decreased K12 mRNA levels compared to control cells. This result indicates an altered differentiation of limbal epithelial cells of aniridia patients. Further studies are necessary to evaluate the mechanism of differentiation of limbal epithelial cells in aniridia.

[Growth Factor Concentration in Keratocyte Supernatant after Incubation with Human Serum In Vitro]. / Wachstumsfaktorenkonzentration im Kulturüberstand von Keratozyten mit humanem Serum in vitro.

PURPOSE: Application of serum eye drops is an alternative treatment option for therapy-resistant corneal epithelial defects. In case of persisting epithelial defects, cytokines, which are secreted from stromal keratocytes, may play a role in epithelial wound healing. Our aim was to analyze fibroblast growth factor basic (FGFb), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and transforming growth factor ß1 (TGF-ß1) concentration in the supernatant of keratocytes, after incubation with human serum (HS). MATERIALS AND METHODS: Serum eye drops of 10 patients were prepared using the standards of the LIONS Eye Bank Saar-Lor-Lux, Trier/Westpfalz, and were stored at - 80 °C. Primary human keratocytes were isolated from human corneoscleral rings using collagenase A (1 mg/ml) (n = 1) and were cultured in DMEM/Ham's culture medium with 10% fetal bovine serum (FBS). Thereafter, keratocyte cultures were incubated in 15 or 30% HS (in DMEM/F14 without FBS) and FGFb, HGF, KGF and TGF-ß1 concentration was determined with an enzyme-linked immunoabsorbent assay (ELISA) from the supernatant of the culture after 24 hours. We used 15 or 30% HS without keratocytes (under the same storage conditions) as controls. RESULTS: HGF concentration was, for both HS concentrations, significantly higher in the supernatant of keratocytes, than in HS controls (without keratocytes) following 24 hours (p < 0.01). FGFb concentration was significantly increased in 30% HS with keratocytes compared to 30% HS without keratocytes after 24 hours (p < 0.01). TGF-ß1 and KGF concentrations remained unchanged through keratocytes. CONCLUSION: HGF and FGFb concentrations increase in the supernatant of keratocytes, 24 hours after incubation with human serum. These concentration changes may play a role in wound healing of epithelial defects.

Arginase activity, urea, and hydroxyproline concentration are reduced in keratoconus keratocytes.

PURPOSE: Keratoconus (KC) is a disease characterized by thinning and deformation of the cornea, but its etiology remains unknown. Seventy percent of the corneal stroma consists of collagen, which is composed of three intertwined polypeptide chains with glycine-hydroxyproline-proline repeats along their sequence. Arginase is a cytoplasmatic enzyme and catalyzes the conversion of arginine to urea and ornithine, which serves as a precursor for the endogenous synthesis of proline and hydroxyproline. The purpose of this study was to analyze arginase activity, as well as collagen and urea formation in normal and KC-keratocytes and to determine the impact of urea on keratocyte viability and proliferation in vitro. METHODS: Primary human keratocytes were isolated by digestion in collagenase (1.0 mg/mL) from surgically removed corneas of eight keratoconus patients and eight normal human corneal buttons and cultured in DMEM/Ham's F12 medium supplemented with 5 % fetal calf serum. Arginase activity and urea concentration were measured in cell-lysates, hydroxyproline concentration in supernatant of cultured keratocytes using colorimetric assay. Cell viability and cell proliferation of cultured keratocytes were assessed after treatment with urea at concentrations up to10 mM for 24 h using assays for metabolic activity and DNA replication. RESULTS: Arginase activity and urea concentration in KC-keratocytes decreased by about 50 % compared to normal keratocytes (p = 0.003 and p = 0.008). Hydroxyproline synthesized by cultured KC-keratocytes was also approximately 50 % less compared to normal keratocytes (p = 0.02) and this difference decreased following treatment with 5.0 or 10.0 mM urea (p = 0.02; 0.03), without any change in cell viability (p > 0.09). However, the urea treatment increased modestly (by 20 %) the proliferation rate of KC-keratocytes (p = 0.04; 0.04; 0.04), without any effect on normal cultured keratocytes (p > 0.09). CONCLUSIONS: We identified suppressed arginase activity in the metabolic program of cultured keratoconus keratocytes. The level of urea, as one product of the enzyme arginase was also decreased. This results in impaired collagen synthesis, evidenced in the culture by reduced hydroxyproline concentration. In addition, our data showed that the other product of the arginase reaction, urea supports the proliferation of KC-keratocytes, without changes in their viability. The metabolic reprogramming of keratoconus keratocytes and its impact on development of a clinically detectable keratoconus disease has to be further analyzed.