Search

Introduction: Congenital aniridia is increasingly recognized as part of a complex syndrome with numerous ocular developmental anomalies and non-ocular systemic manifestations. This requires comprehensive care and treatment of affected patients. Our purpose was to analyze systemic diseases in patients with congenital aniridia within the Homburg Aniridia Registry., Methods: Our retrospective, monocentric study included patients who underwent a comprehensive ophthalmic examination at Saarland University Medical Center beginning in June 2003. Age, gender, genetic test results, and information on systemic anomalies were recorded. In addition, parents and affected patients were interviewed about developmental and other disease-related conditions., Results: Data from 337 patients (mean age 22 ± 20 [0.3-90] years; 181 women [53.7%]) were analyzed. Genetic testing was performed in 187 (55.5%) patients. A PAX6 mutation was detected in 174 of 187 (93%) cases, of which 20 (10.7%) had WAGR(O) syndrome. Systemic diseases were detected in 155 of 337 (46%) patients, with the most common being obesity (29 [8.6%]), thyroid disease (28 [8.3%]), hypertension (26 [7.7%]), intellectual disability (22 [6.5%]), diabetes mellitus (19 [5.6%]), auditory perception disorder/speech development delay (16 [4.7%]), and epilepsy (12 [3.6%])., Conclusions: A comprehensive analysis of patients with aniridia and systemic effects reveals the complexity of this rare disorder, which goes beyond ocular symptoms and can have profound effects on metabolic balance, cardiovascular health, and the central nervous system. Therefore, early genetic diagnosis, early systemic checkup, and adequate treatment, as well as cooperation with pediatrists, neurologists, and audiologists, is suggested in congenital aniridia, which should be considered a syndrome and not an isolated ocular disease., Competing Interests: Declarations. Conflict of Interest: Jessica Obst, Fabian Norbert Fries, Maryam Amini, Annamária Náray, Cristian Munteanu, Tanja Stachon, Shweta Suiwal, Neil Lagali, Berthold Seitz, Barbara Käsmann-Kellner, and Nóra Szentmáry have no conflict of interests to declare. Nóra Szentmáry is an Editorial Board member of Ophthalmology and Therapy. Nóra Szentmáry was not involved in the selection of peer reviewers for the manuscript nor any of the subsequent editorial decisions. Ethical Approval: This study was approved by the Ethics Committee of Saarland (No. 144/15) and complied with the provisions of the Declaration of Helsinki and its amendments., (© 2025. The Author(s).)

Purpose: Rose Bengal Photodynamic Therapy (RB-PDT) offers dual therapeutic benefits by enhancing corneal stiffness and providing antibacterial activity, presenting significant potential for patients with keratoconus complicated by keratitis. Our purpose was to assess the effect of rose bengal photodynamic therapy (RB-PDT) on the expression of pro-inflammatory cytokines and chemokines, as well as on extracellular matrix (ECM)-related molecules, in lipopolysaccharide (LPS)-induced inflammation of keratoconus human corneal fibroblasts (KC-HCFs). Additionally, the involvement of the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways which are downstream of the Toll-like receptor 4 (TLR4) pathway were examined., Methods: KC-HCFs were stimulated with varying concentrations of LPS (0-10 μg/ml), which was followed by RB-PDT. The expression levels of interleukin-1β (IL-1β), IL-6, IL-8, interferon alpha 2 (IFNA2), IFNB1, intercellular adhesion molecule 1 (ICAM-1), chemokine (C-C motif) ligand 4 (CCL-4), collagen I, collagen V, lysyl oxidase (LOX), transforming growth factor β 1(TGF-β1) were measured using qPCR, ELISA, or western blot. The activation of the NF-κB and MAPK pathways was assessed using qPCR and western blot., Results: In LPS-induced inflammation of KC-HCFs, the expression of IL-6 was further amplified by the treatment with RB-PDT (p = 0.001). However, the activation of the MAPK and NF-κB pathways did not increase following RB-PDT. Additionally, RB-PDT reduced the transcription of collagen I and collagen V (p≤0.03), while the transcription of LOX and TGF-β1 secretion remained unchanged in KC-HCFs exposed to LPS., Conclusion: In LPS-induced inflammation of KC-HCFs treated with RB-PDT, despite the increased expression of pro-inflammatory cytokines, the activation of the TLR4 signaling pathways is lacking. RB-PDT may have no adverse effects on corneal scar formation of keratoconus corneas in the short term., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Chai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: Our aim was to examine the expression of PAX6 and keratocyte-specific markers in human limbal stromal cells (LSCs) in congenital aniridia (AN) and in healthy corneas, in vitro ., Methods: Primary human LSCs were extracted from individuals with aniridia (AN-LSCs) ( n  = 8) and from healthy corneas (LSCs) ( n  = 8). The cells were cultured in either normal-glucose serum-containing cell culture medium (NGSC-medium) or low-glucose serum-free cell culture medium (LGSF-medium). Analysis of PAX6 and keratocyte-specific markers was conducted using qPCR and Western blotting. The keratocyte-specific markers included Collagen I (COL1A1), Collagen III (COL3A1), Collagen V (COL5A1), α-smooth muscle actin (ACTA2), Aldehyde Dehydrogenase 3 Family, Member A1 (ALDH3A1), Keratocan (KER), Lumican (LUM), and CD34., Results: PAX6 mRNA expression exhibited a significant decrease in AN-LSCs compared to LSCs in both NGSC- and LGSF-medium ( p  = 0.04; p  = 0.014). There was a marked reduction in COL5A1 mRNA expression ( p  = 0.011), accompanied by notably higher ALDH3A1 and KER mRNA levels ( p  = 0.007; p  = 0.013) in AN-LSCs compared to LSCs when using NGSC-medium. In LGSF-medium, AN-LSCs showed a significant increase in COL1A1 and COL5A1 mRNA expression compared to LSCs ( p  = 0.048; p  = 0.002). Moreover, COL1A1 and α-SMA protein expression were significantly elevated in AN-LSCs compared to LSCs in LGSF-medium ( p  = 0.048, p  = 0.008)., Conclusions: Our investigation affirms the altered expression of PAX6 and keratocyte-specific markers in AN-LSCs relative to healthy controls. Both NGSC- and LGSF-medium exerted distinct effects on both LSCs and AN-LSCs. The observed variations in PAX6 and keratocyte-specific marker expression in AN-LSCs may play a pivotal role in the development and progression of aniridia-associated keratopathy.

Purpose: This study evaluates the microRNA (miRNA) expression profile in primary limbal epithelial cells (pLECs) of patients with aniridia., Methods: Primary human LECs were sampled and isolated from 10 patients with aniridia and 10 healthy donors. The miRNA profile was analyzed using miRNA microarrays. The biological roles of miRNA-validated target genes were delineated in silico by the enrichment analyses of the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The expression of the most deregulated miRNAs was analyzed using quantitative real-time PCR (qRT-PCR)., Results: Microarray analysis revealed 10 differentially expressed miRNAs in pLECs of patients with aniridia relative to healthy controls (fold change = ≤ -2 or ≥ +2), nevertheless these were only differentially expressed using an unadjusted P value < 0.05. The qRT-PCR validation confirmed the significantly altered expression of miR-138-5p in pLECs of patients with aniridia (P = 0.005). In silico GO analysis of miR-138-5p target genes revealed the potential biological functions of miR-138-5p in regulating various cellular and molecular processes, including the positive regulation of cell motility, G1/S phase cell cycle transition, and cell migration, as well as the negative role in regulating epithelial cell differentiation. Pathway analysis highlighted the main involvement of the PI3K-Akt, Hippo, Wnt, Focal adhesion, cAMP, p53, IL-17, Jak-STAT, and MAPK-signaling pathways., Conclusions: This study revealed miRNA expression profile in pLECs of patients with aniridia using miRNA microarray and identified miRNAs that had not been previously reported for aniridia LECs. Our study also provides functional and pathway information that can be used to predict possible mechanism of miRNA function in LECs, thereby bridging the gap in the pathogenesis of AAK studies.

To determine the impact of increased glucose concentration on gene expression of primary healthy human limbal fibroblasts (LFCs) and congenital aniridia human limbal fibroblasts (AN-LFCs), in vitro. LFCs (n = 8) and AN-LFCs (n = 8) were isolated and cultured in serum containing DMEM, including either normal glucose (17.5 mM) or increased glucose (70 mM) concentration for 48h or 72h, respectively. mRNA and protein expression of transforming growth factor beta 1 (TGF-β1), alpha-smooth muscle actin (ACTA)2A1, SMAD 2/3, hypoxia markers such as nuclear factor kappa B (NFκB), inducible nitric oxide synthase (iNOS), hypoxia-inducible factor 1-alpha (HIF-1ɑ), oxidative stress markers such as nuclear factor erythroid 2-related factor 2 (Nrf2) and Catalase (CAT) were analyzed using qPCR and Western blot. In 70 mM glucose concentration medium for 48 h, TGF-β1 mRNA expression was significantly lower (p = 0.001, p < 0.001), Nrf2 (p = 0.001, p = 0.001) and CAT (p = 0.001, p = 0.001) mRNA expression was significantly higher in LFCs and AN-LFCs, than using 17.5 mM glucose concentration medium. In addition, in 70 mM glucose concentration medium for 48 h, SMAD 2, SMAD 3, NFκB, HIF-1ɑ mRNA expression was significantly lower in AN-LFCs, than in 17.5 mM glucose concentration medium (p = 0.003, p = 0.002, p = 0.008, p = 0.020). At this time-point in 70 mM glucose concentration medium, at protein level, TGF-β1, SMAD2/3 and NFκB were significantly lower in AN-LFCs, than in 17.5 mM glucose concentration medium (p = 0.041, p = 0.002, p = 0.012). In 70 mM glucose concentration medium for 72h, TGF-β1 was significantly higher (p < 0.001, p < 0.001) and Nrf2 (p = 0.001, p = 0.001) and CAT (p < 0.001, p < 0.001) mRNA were significantly lower in LFCs and AN-LFCs, than in 17.5 mM glucose concentration medium. At this time-point, in 70 mM glucose concentration medium, NFκB mRNA was significantly higher (p < 0.001) in LFCs, than in 17.5 mM glucose concentration DMEM medium. In 70 mM glucose concentration medium for 72 h, TGF-β1 and NFκB protein were significantly lower in AN-LFCs, than in 17.5 mM glucose concentration medium (p < 0.001, p < 0.001). Our study confirmed that high glucose concentration has an impact on TGF-β1 and NFκB signaling both in AN-LFCs and LFCs. These findings highlight that prolonged exposure to high glucose levels may contribute to cellular stress and dysfunction in LFCs and AN-LFCs., (Copyright © 2024. Published by Elsevier Ltd.)

Introduction: Congenital aniridia is a rare panocular disorder that is associated with varying degrees of impairment of visual acuity. The COST Action (CA18116) developed a survey (aniridia-net.eu) to assess patient-reported experiences with congenital aniridia and its impacts on vision and daily life. Here, we correlate the survey responses of German patients with congenital aniridia with clinical ophthalmology data acquired at the Homburger Aniridia Center., Patients and Methods: The patients completed the German-language version of a 20-point ANIRIDIA-NET survey. The survey included demographic information, the most common symptoms caused by the disease, difficulties caused by visual impairment in various life situations, and the frequency of using visual aids in daily life. As for clinical data, best-corrected visual acuity (BCVA) as well as corneal, lens, and glaucoma status were collected., Results: A total of 71 participants, 27 (38.0%) children and 44 (61.7%) adults, completed the questionnaire, with an age range of 28.8 ± 20.2 years (6 - 78 years). Among them, 55 (77.4%) reported daily light sensitivity, 34 (47.8%) experienced dry eyes, 17 (23.9%) had fluctuating vision, 11 (15.4%) reported eye pain, and 5 (7.0%) experienced daily watering eyes. Older patients reported significantly more eye complaints than children (p < 0,001). Notably, patients with more advanced aniridia-associated keratopathy (AAK) exhibited a discernibly lower quality of life (ρ = 0.28, p = 0.027). Similarly, cataract surgery early in life was associated with a more pronounced decline in quality of life (ρ = - 0.36, p = 0.002). Thirty-five (49.2%) patients never needed assistance for their commute to school/work, 27 (38.0%) and 22 (30.9%) never needed assistance for their daily routines at home or various social activities, respectively. Regarding the use of visual aids, 39 (24.9%) reported that they always used visual aids at work or school, 24 (33.8%) during social activities, and 32 (45.1%) during free time activities., Conclusions: Although congenital aniridia is associated with reduced visual acuity, the majority of affected individuals, especially during childhood, report that they were able to manage personal communication and various life situations independently and without significant difficulties, despite their eye-related issues. Visual aids serve as crucial support for them during their transition into adulthood and as they age. Symptoms of congenital aniridia subjects, described by the ANIRIDIA-NET survey, correlated well with clinical findings. Therefore, the questionnaire may provide important information for the treating ophthalmologist for follow-up examination of these patients and improvement in their life quality., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Purpose: The potential risks and benefits of cataract surgery, in context of congenital aniridia (CA), are not widely understood, yet. Our purpose was to investigate the effect of lens properties on visual acuity (VA), aniridia-associated keratopathy (AAK) stage and presence of glaucoma at the Homburg Aniridia Center., Methods: CA subjects, examined at the Department of Ophthalmology of Saarland University between June 2003 and January 2022, were included. VA, slit-lamp examination, AAK grade, and glaucoma evaluation data were extracted from the medical records, from the first visit to the center. Eyes were categorized as clear lens, cataract, pseudophakic, aphakic, or subluxated lens. Patients were grouped by age (0-10, 10-20, 20-40, 40+ years)., Results: In 553 eyes of 286 CA subjects (age 19.9 ± 19.9 (0-83) years, 46.1% males), analysis revealed significant differences in VA and mean IOP (ANOVA p  < 0.0001; p  = 0.001, respectively) with lens status. Lens status was strongly associated with AAK Grade and glaucoma presence ( p  < 0.0001 for both). In age subgroups, AAK Stage was strongly associated with lens status in the 0-10 years ( p  < 0.001), 10-20 years ( p  < 0.001), and 40+ years ( p  = 0.02) groups and lens status was strongly associated with glaucoma presence in the 0-10 years ( p  = 0.003) and 20-40 years ( p  = 0.002) groups. AAK Stage was the most advanced in pseudophakic and aphakic eyes and presence of glaucoma was more pronounced in pseudophakic, aphakic and subluxated lens eyes., Conclusions: In a large population of CA, previous cataract surgery was associated with higher AAK Grade and presence of secondary glaucoma both in postoperatively pseudophakic and aphakic eyes. Our data indicate that caution is warranted with cataract surgery in congenital aniridia.

Congenital aniridia is a rare eye disease characterized by loss of PAX6 protein leading to aniridia-associated keratopathy that significantly reduces vision. The miR-204-5p is a possible regulator of PAX6 function and here we evaluate its effect in multiple in vitro and in vivo models. In vitro, miR-204-5p overexpression suppressed vascular factor ANGPT1 in human limbal stem cells (T-LSC) and Pax6-knockdown LSC (mut-LSC), and in primary human limbal epithelial cells (LEC) at the gene and protein levels and following LPS stimulation. However, miR-204-5p inhibited VEGFA expression only in mut-LSCs and LPS-stimulated LEC. Also, miR-204-5p increased PAX6 expression in mut-LSC and differentiated corneal epithelial cells, but not in LEC. Topical miR-204-5p after LPS-induced keratitis in mice failed to suppress Vegfa, Angpt1, Il-1β, and Tnf-α or rescue Pax6 levels in contrast to in vitro results, although it significantly reduced the inflammatory infiltrate in the cornea. In Pax6 Sey/+ aniridia mice, miR-204-5p did not rescue PAX6 levels or suppress Vegfa, Angpt1, or inhibit the ERK1/2 pathway. While short-term miR-204-5p treatment effectively suppresses VEGFA and ANGPT1 and enhances PAX6 expression in multiple corneal epithelia, effects are variable across primary and immortalized cells. Effects of longer-term in vivo treatment, however, require further study., (© 2024. The Author(s).)

Aniridia-associated keratopathy originates from a haploinsufficiency of the transcription factor PAX6 (PAX6 +/- ). In the corneal epithelium of PAX6 +/- mice, a significant increase in oxidized proteins was observed, accompanied by impaired compensation for elevated oxidative stress (OS). The extent to which limbal fibroblast cells (LFCs) are affected by an increased susceptibility to OS in cases of congenital aniridia (AN) has not been determined, yet. Our aim was to examine the impact of OS on antioxidant enzyme expression in normal and AN-LFCs. Following isolation and culture of primary LFCs (n = 8) and AN-LFCs (n = 8), cells were treated with cobalt chloride for 48 h to chemically induce hypoxic conditions and OS. Subsequently, HIF-1α/-2α, PHD1/2, Nrf2, CAT, SOD1, PRDX6, and GPX1 gene expression was examined by qPCR. SOD1, PRDX6, and GPX1 protein levels were assessed from the cell lysate by Western blot. The induction of hypoxia led to reduced HIF-1α gene expression in both fibroblast groups (p≤0.008), while the decrease in PHD1 was limited to AN-LFCs (p = 0.0007). On the other hand, under hypoxic conditions, PHD2 showed higher mRNA expression in AN-LFCs compared to normal LFCs (p = 0.013). As a result of OS, the mRNA levels of Nrf2 (p<0.0001) and the antioxidant enzymes CAT (p = 0.005), SOD1 (p = 0.005), GPX1 (p = 0.002) decreased in AN-LFCs. This was accompanied by an increased protein expression of SOD1 (p = 0.019) and PRDX6 (p=0.0009). In the normal LFC group, the induced extent of OS had no impact on the gene (p≥0.151) and protein expression (p ≥ 0.629) of antioxidant enzymes, except for the GPX1 mRNA level (p = 0.027). AN-LFCs exhibit higher susceptibility to OS than normal LFCs. Therefore, in AN-LFCs, there are sustained alterations in gene and protein expression of antioxidative enzymes even after 48 h of CoCl 2 treatment., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: In congenital aniridia caused by mutations in paired box 6 (PAX6), PAX6 influences the migration and differentiation of limbal epithelial cells (LECs), thereby playing a pivotal role in aniridia-associated keratopathy. The antidepressants ritanserin and duloxetine affect PAX6 expression in LECs. Limbal stromal cells, which support limbal epithelial stem cells, are crucial in the limbal stem cell niche. This study explores how ritanserin and duloxetine influence gene expression in primary human limbal stromal cells from subjects with congenital aniridia and from healthy subjects, in vitro., Methods: Primary human limbal stromal cells from corneas affected by aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8) were isolated and cultured in either low-glucose serum-free (LGSF) or normal-glucose serum-containing (NGSC) media. Cells were treated with 4 µM ritanserin or duloxetine for 24 h. Quantitative PCR (qPCR) and western blot were used to assess the expression of PAX6, FOSL2, TGF-β1, ACTA2A1, LUM, COL1A1, COL5A1, DSG1, FABP5 and ADH7., Results: In AN-LSCs with LGSF-medium, ritanserin increased PAX6 messenger RNA (mRNA) (p = 0.007) and decreased TGF-β1 and FOSL2 mRNA levels (P = 0.005, P = 0.038). In addition, TGF-β1 protein levels decreased with both treatments (P = 0.02, P = 0.007), and FABP5 protein level increased, using ritanserin (P = 0.019). In LSCs with LGSF-medium, ACTA2A1 mRNA levels decreased using ritanserin and duloxetine (P = 0.028; P = 0.031), while FABP5 mRNA levels increased with ritanserin treatment (P = 0.003). Also, duloxetine use reduced α-SMA protein (P = 0.013) and increased FABP5 protein levels (P = 0.029). In LSCs with NGSC-medium, ritanserin elevated LUM, FABP5 and ADH7 mRNA and protein levels (P = 0.025, P = 0.003, P = 0.047, P = 0.024, P = 0.013, P = 0.039)., Conclusions: The results of our study confirmed that the antipsychotropic drugs ritanserin and duloxetine alter PAX6 and TGF-β1 gene expression in AN-LSCs cultured in LGSF-medium. These drugs were found to have an impact on retinoic acid signaling pathways and keratocyte characteristic markers both in LSCs and AN-LSCs, using different culture media., (© 2024. The Author(s).)

Purpose: To examine the in-vitro expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in corneal stromal cells by distinguishing between fibroblasts and keratocytes of healthy and keratoconus (KC) corneas., Methods: Stromal cells were isolated from healthy and KC corneas (n = 8). A normal-glucose, serum-containing cell culture medium (NGSC-medium) was used for cultivation of healthy human corneal fibroblasts (HCFs) and KC human corneal fibroblasts (KC-HCFs). In order to obtain a keratocyte phenotype, the initial cultivation with NGSC-medium was changed to a low-glucose, serum-free cell culture medium for healthy (Keratocytes) and KC cells (KC-Keratocytes). Gene and protein expression of MMP-1, -2, -3, -7, -9 and TIMP-1, -2, -3 were measured by quantitative PCR and Enzyme-Linked Immunosorbent Assay (ELISA) from the cell culture supernatant., Results: KC-HCFs demonstrated a lower mRNA gene expression for MMP-2 compared to HCFs. In contrast to their respective fibroblast groups (either HCFs or KC-HCFs), Keratocytes showed a higher mRNA gene expression of TIMP-3, whereas TIMP-1 mRNA gene expression was lower in Keratocytes and KC-Keratocytes. Protein analysis of the cell culture supernatant revealed lower concentrations of MMP-1 in KC-HCFs compared to HCFs. Compared to Keratocytes, TIMP-1 concentrations was lower in the cell culture supernatant of KC-Keratocytes. In HCFs and KC-HCFs, protein levels of MMP-1 and TIMP-1 were higher and MMP-2 was lower compared to Keratocytes and KC-Keratocytes, respectively., Conclusion: This study indicates an imbalance in MMP and TIMP expression between healthy and diseased cells. Furthermore, differences in the expression of MMPs and TIMPs exist between corneal fibroblasts and keratocytes, which could influence the specific proteolytic metabolism in-vivo and contribute to the progression of KC., (© 2024. The Author(s).)

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/cm 2 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/cm 2 fluence in HCFs (p < 0.001) and KC-HCFs (p < 0.0001), and from 0.35 J/cm 2 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/cm 2 fluence in T-LSCs (p < 0.0001), HCE-T (p < 0.05), and KC-HCFs (p < 0.001) and from 0.17 J/cm 2 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., Competing Interests: The authors declare that there is no conflict of interest., (Thieme. All rights reserved.)

Purpose: To investigate the effect of rose bengal photodynamic therapy on lipopolysaccharide-induced inflammation in human corneal fibroblasts. Furthermore, to analyze potential involvement of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways in this process., Methods: Human corneal fibroblast cultures underwent 0-2.0 µg/mL lipopolysaccharide treatment, and 24 h later rose bengal photodynamic therapy (0.001% RB, 565 nm wavelength illumination, 0.17 J/cm 2 fluence). Interleukin-6, interleukin-8, intercellular adhesion molecule-1, interferon regulatory factor-3, interferon α2, and interferon β1 gene expressions were determined by quantitative PCR. Interleukin-6, interleukin-8, and C-C motif chemokine ligand-4 concentrations in the cell culture supernatant were measured by enzyme-linked immunosorbent assays and intercellular adhesion molecule-1 protein level in human corneal fibroblasts by western blot. In addition, the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways were investigated by quantitative PCR and phosphorylation of nuclear factor kappa B p65 and p38 mitogen-activated protein kinase by western blot., Results: Rose bengal photodynamic therapy in 2.0 µg/mL lipopolysaccharide-stimulated human corneal fibroblasts triggered interleukin-6 and interleukin-8 mRNA ( p  < .0001) and interleukin-6 protein increase ( p  < .0001), and downregulated intercellular adhesion molecule-1 expression ( p  < .001). C-C motif chemokine ligand-4, interferon regulatory factor-3, interferon α2, and interferon β1 expressions remained unchanged ( p  ≥ .2). Rose bengal photodynamic therapy increased IκB kinase subunit beta, nuclear factor kappa B p65, extracellular signal-regulated kinases-2, c-Jun amino terminal kinase, and p38 transcription ( p  ≤ .01), and triggered nuclear factor kappa B p65 and p38 mitogen-activated protein kinase phosphorylation ( p  ≤ .04) in lipopolysaccharide treated human corneal fibroblasts., Conclusion: Rose bengal photodynamic therapy of lipopolysaccharide-stimulated human corneal fibroblasts can modify the inflammatory response by inducing interleukin-6 and interleukin-8 expression, and decreasing intercellular adhesion molecule-1 production. C-C motif chemokine ligand-4, interferon regulatory factor-3, and interferon α and β expressions are not affected by rose bengal photodynamic therapy in these cells. The underlying mechanisms may be associated with nuclear factor kappa B and p38 mitogen-activated protein kinase pathway activation.

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% CO 2 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 ., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Purpose: To investigate expression of cytokines and ROS-related genes in stromal cells of healthy and keratoconus (KC) corneas., Methods: Expression analysis was performed for cytokines including several interleukins (IL), Tumor necrosis factor-α (TNF-α), Transforming growth factor-β1 (TGF-β1), Interferon-γ (IFN-γ) and ROS-related genes such as Catalase, Glutathione peroxidase 1, NADPH oxidase 1, superoxide dismutase 1 in corneal fibroblasts (HCFs/KC-HCFs) or keratocytes (Keratocytes/KC-Keratocytes) by qPCR and ELISA., Results: Gene and protein expression of most inflammatory markers was decreased in keratocytes compared to fibroblasts, whereas no differences were found between healthy and keratoconus cells for the majority of cytokines measured. TNF-α expression was increased at gene (KC keratocytes) and protein levels (supernatant of Keratocytes/KC-Keratocytes) compared to corneal fibroblasts. No differential expression of ROS-related genes was detected between healthy and diseased cells in both fibroblasts and keratocytes., Conclusion: Increased expression of several inflammatory markers described as altered in KC was not evident in KC cells in vitro .

Purpose: Congenital aniridia is a rare disease, which is in most cases related to PAX6 haploinsufficiency. Aniridia associated keratopathy (AAK) also belongs to ocular signs of congenital aniridia. In AAK, there is corneal epithelial thinning, corneal inflammation, vascularization and scarring. In advanced stage AAK, typically, conjunctival epithelial cells slowly replace the corneal epithelium. Based on previous results we hypothesize that alterations of the conjunctival cells in congenital aniridia may also support the corneal conjunctivalization process. The aim of this study was to identify deregulated proteins in conjunctival impression cytology samples of congenital aniridia subjects., Methods: Conjunctival impression cytology samples of eight patients with congenital aniridia [age 34.5 ± 9.9 (17-51) years, 50% female] and eight healthy subjects [age 34.1 ± 11.9 (15-54) years, 50% female] were collected and analysed using mass spectrometry. Proteomic profiles were analysed in terms of molecular functions, biological processes, cellular components and pathway enrichment using the protein annotation of the evolutionary relationship (PANTHER) classification system., Results: In total, 3323 proteins could be verified and there were 127 deregulated proteins (p < 0.01) in congenital aniridia. From the 127 deregulated proteins (DEPs), 82 altered biological processes, 63 deregulated cellular components, 27 significantly altered molecular functions and 31 enriched signalling pathways were identified. Pathological alteration of the biological processes and molecular functions of retinol binding and retinoic acid biosynthesis, as well as lipid metabolism and apoptosis related pathways could be demonstrated., Conclusions: Protein profile of conjunctival impression cytology samples of aniridia subjects identifies alterations of retinol binding, retinoic acid biosynthesis, lipid metabolism and apoptosis related pathways. Whether these changes are directly related to PAX6 haploinsufficiency, must be investigated in further studies. These new findings offer the possibility to identify potential new drug targets., (© 2023 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Purpose: Aniridia is a rare corneal disease that is often associated with aniridia-associated keratopathy (AAK). In AAK, the conjunctival tissue crosses the limbal border, forming a corneal pannus that extends into the corneal center. With increasing AAK severity, corneal pannus formation, vascularization, and ocular surface inflammation increase. The purpose of this study was to investigate inflammation-related mRNA expression in conjunctival epithelial cells in AAK and its relationship with AAK severity., Methods: Using impression cytology, bulbar conjunctival cells were sampled from 20 subjects with congenital aniridia and 20 age-matched and sex-matched healthy control subjects. RNA was extracted, and mRNA analyses were performed using microarray, which was evaluated for inflammatory markers., Results: In the analyzed aniridia subjects, 70 deregulated mRNAs encoding proinflammatory or antiinflammatory cytokines or factors associated with chronic inflammation, including increased IL-1, IL-8, and MIP3A/CCL20 mRNA. The most downregulated mRNA was TIMP3, and the most upregulated mRNA was Protein c-Fos.Of the 70 mRNAs, 14 inflammation-related genes were altered only in the mild AAK forms, whereas only 2 mRNAs were altered only in the severe AAK forms (TLR4 and PPARG)., Conclusions: The expression of numerous proinflammatory and antiinflammatory cytokines is deregulated at the ocular surface of aniridia subjects with mild AAK. Thus, early antiinflammatory treatment may prevent or slow down corneal scarring and pannus formation in aniridia subjects., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: This study analysed the causative factors and clinical characteristics of acute and chronic ocular sequelae of Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) treated at a large third-referral centre in a developed country (Hungary) over a 15-year period., Methods: This was a retrospective review of patients with acute and/or chronic SJS/TEN who were managed between 2006 and 2020 at the Department of Ophthalmology of Semmelweis University in Budapest, Hungary. For each subject, clinical data, including patient demographics, clinical history, causative agents of SJS/TEN, and conservative and surgical treatment details, were reviewed., Results: Ninety-six eyes of 48 patients were included (28 female; 58.3%); the age at disease onset was 32.1 ± 22.4 years. The most common causative factors were medicines (n = 36; 75.0%). Among these drugs, 29.2% were nonsteroidal anti-inflammatory drugs (NSAIDs) (n = 14), 20.8% were antibiotics (n = 10) and 14.6% were antiepileptic drugs (n = 7). In patients with chronic SJS/TEN, the most commonly found ocular sequelae were conjunctival hyperaemia in 45 (56.3%) eyes, symblepharon in 38 (47.5%) eyes, trichiasis/distichiasis in 37 (46.3%) eyes, corneal neovascularization in 31 (38.8%) eyes and corneal scarring in 29 (36.3%) eyes. In patients with chronic SJS/TEN, the most frequently used topical conservative treatment included antibiotics in 53 (66.3%) eyes, preservative-free artificial tears in 50 (62.5%) eyes and topical corticosteroids in 42 (52.5%) eyes of 40 patients. The most frequently performed ocular surgeries for managing chronic ocular sequelae in patients with SJS/TEN were epilation for trichiasis (n = 27; 33.8%), cataract surgery (n = 14; 17.5%), entropion surgery (n = 12; 15.0%), penetrating keratoplasty (PK) (n = 11; 13.8%) and amniotic membrane transplantation (n = 4; 5.0%)., Conclusion: Our results suggest that NSAIDs, antibiotics and antiepileptic drugs are the most common causative factors for SJS/TEN in Hungary. Like in other countries, in Hungary, the ocular management of patients with acute and chronic SJS/TEN is heterogeneous, and most cases do not follow modern therapeutic guidelines., (© 2024. The Author(s).)

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., Competing Interests: The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/).)

Purpose: To investigate collagen I, collagen V, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), lysyl oxidase (LOX), transforming growth factor β1 (TGF-β1) and interleukin-6 (IL-6) expression in healthy and keratoconus human corneal fibroblasts (HCFs and KC-HCFs), 24 h after Rose Bengal photodynamic therapy (RB-PDT)., Methods: HCFs were isolated from healthy human corneal donors ( n  = 5) and KC-HCFs from elective penetrating keratoplasties ( n  = 5). Both cell cultures underwent RB-PDT (0.001% RB concentration, 0.17 J/cm 2 fluence) and 24 h later collagen I, collagen V, NF-κB, LOX, TGF-β1 and IL-6 mRNA and protein expression have been determined using qPCR and Western blot, IL-6 concentration in the cell culture supernatant by ELISA., Results: TGF-β1 mRNA expression was significantly lower ( p  = 0.02) and IL-6 mRNA expression was significantly higher in RB-PDT treated HCFs ( p  = 0.01), than in HCF controls. COL1A1, COL5A1 and TGF-β1 mRNA expression was significantly lower ( p  = 0.04; p  = 0.02 and p  = 0.003) and IL-6 mRNA expression was significantly higher ( p  = 0.02) in treated KC-HCFs, than in KC-HCF controls. TGF-β1 protein expression in treated HCFs was significantly higher than in HCF controls ( p  = 0.04). IL-6 protein concentration in the HCF and KC-HCF culture supernatant after RB-PDT was significantly higher than in controls ( p  = 0.02; p  = 0.01). No other analyzed mRNA and protein expression differed significantly between the RB-PDT treated and untreated groups., Conclusions: Our study demonstrates that RB-PDT reduces collagen I, collagen V and TGF-β1 mRNA expression, while increasing IL-6 mRNA and protein expression in KC-HCFs. In HCFs, RB-PDT increases TGF-β1 and IL-6 protein level after 24 h.

Purpose: To investigate human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium (CM)., Methods: A human corneal epithelial cell line (HCE-T), human corneal fibroblasts (HCF) and keratoconus fibroblasts (KC-HCF) have been used. Twenty-four hours after RB-PDT (0.001% RB concentration, 565 nm wavelength illumination, 0.17 J/cm2 fluence) cell migration rate using scratch assay and growth factor concentrations in the cell culture supernatant using ELISA have been determined. In addition, the effect of CM has been observed., Results: RB-PDT significantly reduced migration rate in all cell types, compared to controls (p≤0.02). Migration rate of HCE-T cultures without RB-PDT (untreated) was significantly higher using HCF CM after RB-PDT, than using HCF CM without RB-PDT (p<0.01). Similarly, untreated HCF displayed a significantly increased migration rate with HCE-T CM after RB-PDT, compared to HCE-T CM without treatment (p<0.01). Furthermore, illumination alone and RB-PDT significantly decreased keratinocyte growth factor (KGF) concentration in HCF and KC-HCF supernatant, and RB-PDT significantly decreased soluble N-Cadherin (SN-Cad) concentration in HCF supernatant, compared to controls (p<0.01 for all). In HCE-T CM, RB-PDT increased hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGFb) concentration (p≤0.02), while decreasing transforming growth factor β (TGF-β) concentration (p<0.01). FGFb concentration increased (p<0.0001) and TGF-β concentration decreased (p<0.0001) in HCF CM, by RB-PDT. Epidermal growth factor (EGF), HGF, and TGF-β concentration decreased (p≤0.03) and FGFb concentration increased (p<0.01) in KC-HCF CM, using RB-PDT., Conclusions: HCE-T, HCF and KC-HCF migration rate is reduced 24 hours after RB-PDT. In contrast, HCE-T migration is enhanced using HCF CM after RB-PDT, and HCF migration rate is increased through HCE-T CM following RB-PDT. Modulation of EGF, KGF, HGF, FGFb, TGF-β and N-Cadherin secretion through RB-PDT may play an important role in corneal wound healing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commecial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Smits et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Aniridia is a rare congenital panocular disease associated with varying degrees of visual acuity impairment., Objective: To assess the experiences of congenital aniridia patients in Hungary, with visual impairment using a questionnaire developed by the ANIRIDIA-NET., Patients and Method: Patients completed the Hungarian version of the 20-item ANIRIDIA-NET questionnaire with our assistance. The questionnaire covered demographic data, the most common complaints caused by the disease, the difficulties caused by low vision in different life situations and the frequency of low vision aids used in daily life., Results: 33 subjects (17 female [51.51%] and 16 male [48.48%]), 16 (48.5%) children and 17 (51.5%) adults completed the questionnaire, with an age of 25.69 ± 17.49 years (5-59 years). Daily photosensitivity was reported by 27 (81.8%), dry eyes by 5 (15.2%), tearing by 4 (12.1%), fluctuating vision by 3 (9.1%), and eye pain by 2 (6.1%) subjects. The majority of respondents said that personal communication with schoolmates (16 [48.5%]) or colleagues at work (11 [33.3%]) never caused difficulties because of their visual impairment. 29 people (87.9%) never needed help with daily routines at home, 24 (72.7%) with getting to school/work and 17 (51.5%) with various activities. 29 people (87.8%) never used low vision aids for communication, 23 (69.7%) for travelling, 20 (60.6%) for participating in social activities, 18 (54.5%) for studying/work., Conclusion: Although aniridia is associated with reduced visual acuity, the majority of people with congenital aniridia, especially in childhood, manage to cope with personal communication and various life situations without difficulty, despite their eye complaints. Low vision aids can be an important aid for them as they grow into adulthood and as they age. Orv Hetil. 2023; 164(34): 1342-1349.

Introduction: Congenital aniridia is a rare disease, characterised by the complete or partial absence of the iris, but lesions may be present in all structures of the eye., Objective: To determine the prevalence of ocular diseases in congenital aniridia by analyzing patients from a Hungarian centre., Patients and Methods: Patients at the Department of Ophthalmology of Semmelweis University, examined between October 2005 and May 2022, have been included. After taking the patients' medical history, a detailed ophthalmological examination has been performed., Results: Of the 82 patients in the database, 33 (age 25.69 ± 17.49 [5-59] years, 17 females [51.51%]) presented for examination and 65 eyes were examined. Nystagmus was found in 45 eyes of 23 patients (69.23%), and the patients' uncorrected distance visual acuity was 0.14 ± 0.128 (0.9 logMAR; 0.63-0.005). The aniridia-associated keratopathy was Grade 0 in 8 eyes (12.3%), Grade 1 in 10 eyes (15.38%), Grade 2 in 16 eyes (24.62%), Grade 3 in 4 eyes (6.15%) and Grade 4 in 25 eyes (38.46%). 30 eyes (46.15%) of 15 patients had secondary glaucoma, 6 eyes (9.2%) of 3 patients were glaucoma suspect. 8 eyes (12.3%) had a clear lens, 44 eyes (67.69%) had cataract, of which 22 (33.84%) were anterior cortical polar cataracts. 13 eyes (20%) were pseudophakic (PCL) and 7 eyes (10.77%) had lens dislocation or zonular insufficiency. Macular hypoplasia was found in 6 eyes of 3 patients (4.6%) and optic nerve head malformation in 2 eyes of 1 patient (3.03%)., Conclusion: The ocular signs of congenital aniridia are aniridia-associated keratopathy, secondary glaucoma, cataract, macular and optic nerve head hypoplasia. Systematic collaboration of different ophthalmological specialties is required for the management and care of all these ocular abnormalities. Orv Hetil. 2023; 164(4): 148-155.

Acanthamoeba keratitis (AK) is a dangerous infectious disease, which is associated with a high risk of blindness for the infected patient, and for which no standard therapy exists thus far. Patients suffering from AK are thus treated, out of necessity, with an off-label therapy, using drugs designed and indicated for other diseases/purposes. Here, we tested the capability of the off-label anti-amoebic drugs chlorhexidine (CH; 0.1%), dibromopropamidine diisethionate (DD; 0.1%), hexamidine diisethionate (HD; 0.1%), miltefosine (MF; 0.0065%), natamycin (NM; 5%), polyhexamethylene biguanide (PHMB; 0.02%), povidone iodine (PVPI; 1%), and propamidine isethionate (PD; 0.1%) to suppress trophozoite formation of Acantamoeba castellanii and Acanthamoeba hatchetti cysts on non-nutrient agar Escherichia coli plates. Of the eight off-label anti-amoebic drugs tested, only PVPI allowed for a complete suppression of trophozoite formation by drug-challenged cysts for all four Acanthamoeba isolates in all five biological replicates. Drugs such as NM, PD, and PHMB repeatedly suppressed trophozoite formation with some, but not all, tested Acanthamoeba isolates, while other drugs such as CH, DD, and MF failed to exert a relevant effect on the excystation capacities of the tested Acanthamoeba isolates in most, if not all, of our repetitions. Our findings suggest that pre-testing of the AK isolate with the non-nutrient agar E. coli plate assay against the anti-amoebic drug intended for treatment should be performed to confirm that the selected drug is cysticidal for the Acanthamoeba isolate.

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/mm 2 ) to the time point 3 months after surgery (1914 ± 164 cells/mm 2 ; p = 0.080) and to the time point after the first suture removal (1886 ± 557 cells/mm 2 ; p = 0.066) and decrease significantly to the time point after the second suture removal (1650 ± 446 cells/mm 2 ; 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., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Purpose: Evaluation of mRNA and microRNA (miRNA) expression in epithelium and stroma of patients with keratoconus., Methods: The epithelium and stroma of eight corneas of eight patients with keratoconus and eight corneas of eight non-keratoconus healthy controls were studied separately. RNA was extracted, and mRNA and miRNA analyses were performed using microarrays. Differentially expressed mRNAs and miRNAs in epithelial and stromal keratoconus samples compared to healthy controls were identified. Selected genes and miRNAs were further validated using RT-qPCR., Results: We discovered 170 epithelial and 1498 stromal deregulated protein-coding mRNAs in KC samples. In addition, in epithelial samples 180 miRNAs and in stromal samples 379 miRNAs were significantly deregulated more than twofold compared to controls. Pathway analysis revealed enrichment of metabolic and axon guidance pathways for epithelial cells and enrichment of metabolic, mitogen-activated protein kinase (MAPK), and focal adhesion pathways for stromal cells., Conclusions: This study demonstrates significant differences in the expression and regulation of mRNAs and miRNAs in the epithelium and stroma of Patients with KC. Also, in addition to the well-known target candidates, we were able to identify further genes and miRNAs that may be associated with keratoconus. Signaling pathways influencing metabolic changes and cell contacts are affected in epithelial and stromal cells of patients with keratoconus.

Purpose: To determine herpes simplex virus (HSV) DNA prevalence and mean cycle threshold of polymerase chain reaction (PCR) in corneal tissue of patients with penetrating keratoplasty (PKP), with (HSK+) and without (HSK-) previous clinical herpetic keratitis history., Methods: Retrospective review of recipient corneal buttons which were explanted through PKP between March 2010 and September 2018 at the Department of Ophthalmology, Saarland University Medical Center in Homburg/Saar, Germany. Corneal tissue samples were analysed by real-time PCR for the presence of HSV DNA. For each subject, clinical data, including patients' demographics and clinical diagnoses, were collected., Results: In total, 2230 corneal samples (age at the time of the surgery 57.3 ± 19.2 years) of 1860 patients were analysed. HSV PCR was positive in 137 (6.1%) corneal samples, with a 30.57 ± 6.01 (range 14-39) mean cycle threshold (Ct) value. Two hundred ninety-eight (13.4%) corneas of 266 patients were clinically HSK+, and 1932 (86.6%) corneas of 1600 patients were clinically HSK-. HSV DNA was detected significantly more frequently (p < 0.0001) in HSK+ corneal samples (108 corneal samples; 36.2%), than in HSK- corneal samples (29 corneal samples; 1.5%). Ct value was significantly lower in HSK+ than in HSK- corneal samples (29.8 ± 5.8 versus 32.6 ± 5.9; p = 0.008)., Conclusion: Our data demonstrate that a positive clinical history of HSK is related to HSV PCR positivity in about every 2.8th patient. In addition, about every 66th explanted corneal tissue is HSV PCR-positive despite the lack of clinical suspicion. These patients may need additional local/systemic antiviral treatment to avoid newly acquired HSK following penetrating keratoplasty., (© 2021 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

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 Ca 2+ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca 2+ . 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 Ca 2+ supplementation. After PAX6 knock down and Ca 2+ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca 2+ supplementation alone. Using our cell model, with Ca 2+ 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 Ca 2+ 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Keratoconus (KC) is a corneal disorder, associated with oxidative stress, hypoxia and as several times discussed, potentially with thyroid gland dysfunction. We aimed to investigate the effect of thyroxine on transforming growth factor β1 (TGF-β1), collagen I and V (Col I and V) expression in human corneal fibroblasts (HCFs) and human keratocytes of KC corneas, in vitro., Methods: Primary human KC-keratocytes and normal keratocytes were isolated and cultured as corneal fibroblasts or keratocytes. The effect of 0.1 µg/ml and 1.0 µg/ml thyroxine on TGF-β1, Col I and Col V expression was investigated by qPCR, Western blot, and ELISA. Proliferation assay was performed using BrdU ELISA to observe the 24h effect of 1.0 µg/ml thyroxine on keratocytes, in vitro., Results: TGFB1 mRNA expression of normal keratocytes increased following 1.0 µg/ml thyroxine stimulation for 24 h ( p = .036), without changes in protein expression. Col I protein expression of KC-HCFs increased following 1.0 µg/ml thyroxine stimulation for 24 h ( p = .0003). Proliferation of normal and KC keratocytes increased following a 7-day growth period and 24 hours thyroxine administration ( p = .018; p = .024)., Conclusions: Thyroxine may affect the Col I protein expression in KC-HCFs, but not in KC keratocytes, in vitro. Thyroxine administration has no effect on TGF-β1, collagen I and V expression of keratoconus keratocytes. Therefore, an increased thyroxine concentration alone seems not to be causally related to the development of keratoconus.

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., (Copyright © 2021. Published by Elsevier Ltd.)

Congenital PAX6-aniridia is a rare panocular disease resulting from limbal stem cell deficiency. In PAX6-aniridia, the downregulation of the retinol-metabolizing enzymes ADH7 (All-trans-retinol dehydrogenase 7) and ALDH1A1/A3 (Retinal dehydrogenase 1, Aldehyde dehydrogenase family 1 member A3) have been described in limbal epithelial cells (LECs) and conjunctival epithelial cells. The aim of this study was to identify the role of retinol derivates in the differentiation of human LEC and its potential impact on aniridia-associated keratopathy development. Human LEC were isolated from healthy donor corneas and were cultured with retinol, retinoic acid, or pan-retinoic acid receptor antagonist (AGN 193109) acting on RARα, β, γ (NR1B1, NR1B2 NR1B3) or were cultured with pan-retinoid X receptor antagonist (UVI 3003) acting on RXR α, β, γ (retinoid X receptor, NR2B1, NR2B2, BR2B3). Using qPCR, differentiation marker and retinoid-/fatty acid metabolism-related mRNA expression was analysed. DSG1 (Desmoglein 1), KRT3 (Keratin 3), and SPINK7 (Serine Peptidase Inhibitor Kazal Type 7) mRNA expression was downregulated when retinoid derivates were used. AGN 193109 treatment led to the upregulation of ADH7, KRT3, and DSG1 mRNA expression and to the downregulation of KRT12 (Keratin 12) and KRT19 (Keratin 19) mRNA expression. Retinol and all-trans retinoic acid affect some transcripts of corneal LEC in a similar way to what has been observed in the LEC of PAX6-aniridia patients with the altered expression of differentiation markers. An elevated concentration of retinol derivatives in LEC or an altered response to retinoids may contribute to this pattern. These initial findings help to explain ocular surface epithelia differentiation disorders in PAX6-aniridia and should be investigated in patient cells or in cell models in the future in more detail.

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., Competing Interests: Die Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

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/mm 2 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: β = - 13.2, p = 0.001; after last suture removal: β = - 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 (β = 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., Competing Interests: The authors declare that they have no conflict of interest./Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

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 (χ 2  = 4.772, p = 0.028), however, there was not a significant difference between the two years for any other diagnosis (χ 2  ≤ 3.564, p ≥ 0.059). From 2019 to 2020, the percentage of acute intravitreal anti-VEGF injections decreased significantly (7.9% vs. 1.3%, χ 2  = 3.985, p = 0.045), but the proportion of other emergency surgeries did not differ between the two years (χ 2  ≤ 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., Competing Interests: Dr. Tóth reported grants from EFOP-3.6.3-VEKOP-16-2017-00009. The work of Dr. Szentmáry at the Dr. Rolf M. Schwiete Center was supported by the Dr. Rolf M. Schwiete Foundation. No conflicting relationship exists for remaining authors., (Thieme. All rights reserved.)

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.

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

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.

Purpose: To evaluate conjunctival cell microRNA (miRNAs) and mRNA expression in relation to observed phenotype of progressive limbal stem cell deficiency in a cohort of subjects with congenital aniridia with known genetic status., Methods: Using impression cytology, bulbar conjunctival cells were sampled from 20 subjects with congenital aniridia and 20 age and sex-matched healthy control subjects. RNA was extracted and miRNA and mRNA analyses were performed using microarrays. Results were related to severity of keratopathy and genetic cause of aniridia., Results: Of 2549 miRNAs, 21 were differentially expressed in aniridia relative to controls (fold change ≤ -1.5 or ≥ +1.5). Among these miR-204-5p, an inhibitor of corneal neovascularization, was downregulated 26.8-fold in severely vascularized corneas. At the mRNA level, 539 transcripts were differentially expressed (fold change ≤ -2 or ≥ +2), among these FOSB and FOS were upregulated 17.5 and 9.7-fold respectively, and JUN by 2.9-fold, all being components of the AP-1 transcription factor complex. Pathway analysis revealed enrichment of PI3K-Akt, MAPK, and Ras signaling pathways in aniridia. For several miRNAs and transcripts regulating retinoic acid metabolism, expression levels correlated with keratopathy severity and genetic status., Conclusion: Strong dysregulation of key factors at the miRNA and mRNA level suggests that the conjunctiva in aniridia is abnormally maintained in a pro-angiogenic and proliferative state, and these changes are expressed in a PAX6 mutation-dependent manner. Additionally, retinoic acid metabolism is disrupted in severe, but not mild forms of the limbal stem cell deficiency in aniridia., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Purpose: In vivo confocal microscopy (IVCM) is a non-invasive imaging technique that allows morphological analysis as a diagnostic approach of the cornea in real time, thus providing a suspected diagnosis of fungal or amoebic keratitis immediately, whereas culture or PCR require several days or even weeks. Since these infections are rare, it is difficult for ophthalmologists to gain the experience necessary to differentiate infection from normal findings or artefacts. The purpose of this project was to establish a simulator, on which physicians could practice as well as acquiring a database of IVCM images of fungal or amoebic keratitis and respective analyses., Patients and Methods: An IVCM simulator was set up with cadaver human corneas, infected with either acanthamoeba, candida or aspergillus. Twenty-one ophthalmologists were trained in IVC microscopy first in a Dry Lab, then practically on the simulator. For evaluation, the participants were asked to fill out a standardized questionnaire, with a pre- and post-course self-assessment., Results: The self-assessed theoretical and practical skills in differentiating infectious from non-infectious keratitis in IVCM significantly increased ( p = 0.0001, p = 0.0002, respectively). The barrier to use this technique decreased ( p = 0.0474)., Conclusion: A very simple protocol based on a model of ex vivo corneal mycotic and amoebic infections can be used to train novices in the structured approach and diagnostic use of IVCM for corneal infections.

Purpose: The purpose of this study was to analyze the concentration-dependent effects of biguanides (polyhexamethylene biguanide [PHMB], chlorhexidine [CH]); diamidines (hexamidine-diisethionate [HD], propamidine-isethionate [PD], dibromopropamidine-diisethionate [DD]); natamycin (NM); miltefosine (MF); povidone iodine (PVPI), and chlorin e6 PDT on Acanthamoeba trophozoites and cysts, in vitro., Methods: Strain 1BU was cultured in peptone-yeast extract-glucose medium. Trophozoites or cysts were cultured in PYG medium containing each agent at 100%, 50%, and 25% of maximum concentration for 2 hours. The percentage of dead trophozoites was determined using a non-radioactive cytotoxicity assay and trypan blue staining. Treated cysts were also maintained on non-nutrient agar Escherichia coli ( E. coli ) plates and observed for 3 weeks., Results: All tested drugs displayed significant cytotoxic effects on 1BU cells based on the biochemical and staining-based viability assays tested. On non-nutrient agar E. coli plates, neither trophozoites nor freshly formed cysts were observed after PHMB, PD, NM, and PVPI treatment, respectively, within 3 weeks. However, CH-, HD-, DD-, and MF-treated cysts could excyst, multiply, and encyst again., Conclusions: The off-label drugs PHMB, PD, NM, and PVPI are under in vitro conditions more effective against strain 1BU than CH, HD, DD, and MF. Our findings also suggest that the non-nutrient agar E. coli plate assay should be considered as method of choice for the in vitro analysis of the treatment efficacy of anti-amoebic agents., Translational Relevance: Ophthalmologists may optimize the treatment regime against Acanthamoeba keratitis by pre-testing the in vitro susceptibilities of the Acanthamoeba strain against drugs of interest with the non-nutrient E. coli agar plate assay., Competing Interests: Disclosure: L. Shi, None; V. Muthukumar, None; T. Stachon, None; L. Latta, None; M.I. Elhawy, None; G. Gunaratnam, None; E. Orosz, None; B. Seitz, None; A.F. Kiderlen, None; M. Bischoff, None; N. Szentmáry, None, (Copyright 2020 The Authors.)

This investigation aimed to identify early corneal marker and conjunctival epithelial differentiation through transcriptional analysis of limbal explant cultures and study early differentiation patterns of known corneal and conjunctival differentiation markers. 2 mm punch biopsies of limbal region were obtained from 6 donors of the Lions Cornea Bank Saar-Lorloux/Trier-Westpfalz. Limbal explants were dissected into corneal and conjunctival biopsy sections. Biopsies were placed with epithelial side down into 12 Wells. As soon as the outgrowing cells had reached confluence, they were harvested. mRNA expression of corneal differentiation markers KRT12, KRT3, DSG1, PAX6, ADH7 and ALDH1A1, conjunctival markers KRT19, KRT13 and stem cell marker ABCG2 were measured via qPCR. KRT12 and PAX6 protein expressions were evaluated using Western Blot. Results suggested that KRT12 mRNA expression was significantly higher in outgrowing cells from the corneal side of the biopsies as in those from the conjunctival side (p = 0.0043). There was no significant difference in mRNA expression of other analyzed markers comparing with marker expression of outgrown cells from both limbal biopsies (p > 0.13). KRT12 and PAX6 Western Blot analysis showed no difference in cells harvested from both sides. In conclusion, KRT12 mRNA might be a marker to measure corneal origin of cells from limbal biopsies with unknown composition of corneal and conjunctival progenitor cells. KRT3, DSG1, PAX6, ADH7, ALDH1A1, KRT19, KRT13 and ABCG2 mRNA as well as KRT12 and PAX6 protein expression could not contribute to differentiate corneal from conjunctival cell identity from limbal biopsies.

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.