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Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and severe thermal or chemical injury are considered severe ocular surface disorders (OSDs) because they affect the entire ocular surface, including corneal and conjunctival epithelial stem cells. In patients with severe OSDs, the long-term prognosis for limbal transplantation is poor, and the related corneal opacity and cicatrization lead to devastating visual impairment. To date, there is no standardized treatment to improve vision in cases with severe OSD. Investigating novel treatment methods for severe OSDs, our group began cultivated oral mucosal epithelial transplantation in 2002 and developed a limbal-supported rigid-type contact lens that can be applied as a nonsurgical treatment. When used in combination, these treatment methods make it possible to successfully restore vision in cases with severe OSDs.

Ocular surface reconstruction (OSR) using tissue-engineered cultivated oral mucosal epithelial cell sheets (COMECS) is a promising newly developed treatment for patients with severe ocular surface disease. Until now, this technique has used exogenic and undefined components such as mouse-derived 3T3 feeder cells and fetal bovine serum. To minimize associated risks of zoonotic infection or transmission of unknown pathogens and so establish a safe and effective protocol for the next generation of treatment modality, we developed a novel technique for the COMECS protocol, using a feeder-free and serum-free (FFSF) culture system. Following this new protocol, COMECS exhibited 4-5 layers of well-stratified and differentiated cells, and we successfully produced functional COMECS that included holoclone-type stem cells. Immunohistochemistry confirmed the presence of markers for cell junction (ZO1, Desmoplakin), basement membrane assembly (Collagen 7, Laminin 5), differentiation (K13, K3), proliferation (Ki67) and stem/progenitor cells (p75) in the FFSF COMECS. When transplanted to the ocular surfaces of rabbits, the tissue survived for up to 2 weeks. This study represents a first step toward assessing the development of functional FFSF COMECS for safe and ideal OSR.

Purpose: The purpose of this study was to clarify the interactions between macrophages (MPs) and RPE cells in coculture systems to investigate the functional plasticity of RPE cells., Methods: Adherent peritoneal cells or murine MP cell line Raw 264.7 was cocultured with primary RPE cells taken from C57BL/6 mice, with or without lipopolysaccharide (LPS) or TNF-α stimulation. The cytokine levels of the culture supernatants (CSs) were then analyzed with the Bio-Plex murine 23-Plex Panel Assay Kit (Bio-Rad Laboratories). Monocyte chemoattractant protein-1 (MCP-1), IL-6, VEGF, and TNF-α in CS were further quantified by ELISA. The expression profiles, in cocultures, of complement-associated genes, TNF-α, and angiogenesis-associated genes were analyzed by quantitative real-time PCR., Results: The production of MCP-1, IL-6, and VEGF was synergistically elevated when primary MPs or RAW264.7 cells and RPE cells were cocultured compared with those derived from sole cultures of MPs and RPE cells. The synergistic effect was confirmed without direct cell contact and was more prominent in the presence of LPS or TNF-α. TNF-α production by MPs was suppressed by RPE cells. Coculture of RPE cells with RAW264.7 cells increased the gene expression of C3, CFB, and VEGF genes, whereas it reduced those of complement regulatory factors CFH, CD59, clusterin, and TNF-α and antiangiogenic pigment epithelium-derived factor (PEDF)., Conclusions: Our findings indicate the presence of ingenious interactions between MPs and RPE cells that forces the inflammation and complement activation in the vicinity of RPE cells under pathologic conditions.

Purpose: To assess the clinical efficacy of cultivated oral mucosal epithelial transplantation (COMET) for the treatment of persistent epithelial defect (PED)., Methods: We treated 10 eyes of nine patients with PED (Stevens-Johnson syndrome: three eyes; thermal/chemical injury: five eyes; ocular cicatricial pemphigoid: two eyes) with COMET at Kyoto Prefectural University of Medicine, Kyoto, Japan from 2002 to 2008., Results: Preoperatively, PED existed on over more than 50% of the corneal surface in seven eyes. Severe ocular surface inflammation with fibrovascular tissue surrounded the PED in all 10 eyes. At 24-weeks postoperative, PED had improved in all cases except 1 in which the patient was unable to return to the hospital (95% CI, 55.5-99.7; Wilcoxon signed-rank test, p = 0.0078). The preoperative median of logarithmic minimum angle of resolution was 1.85 (range 0.15-2.70), and 1.85, 1.85, and 1.52 at the 4th, 12th, and 24th postoperative week, respectively. The mean total preoperative ocular surface grading score was 7.0 (range 4-17). At 4 and 12 weeks postoperative, the total ocular surface grading score had improved significantly (p = 0.0020, p = 0.0078), and at 24 weeks postoperative, it was 3.0 (range 2-12, p = 0.0234). During the follow-up period (median 23.3 months, range 5.6-39.7 months), no recurrence of PED was observed in any eye, and long-term ocular surface stability was obtained., Conclusion: COMET enabled complete epithelialization of PED and stabilization of the ocular surface in patients with severe ocular surface disease, thus preventing end-stage cicatrization and vision loss at a later stage., (© 2014 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Purpose: We previously documented that ocular surface epithelial cells could regulate ocular surface inflammation and suggested that, while Toll-like receptor 3 upregulates, EP3, one of the prostaglandin E2 receptors, downregulates ocular surface inflammation. Others reported that rebamipide, a gastroprotective drug, could not only increase the gastric mucus production, but also suppressed gastric mucosal inflammation and that it was dominantly distributed in mucosal tissues. The eyedrop form of rebamipide, approved in Japan for use in the treatment of dry eye diseases, upregulates mucin secretion and production, thereby suppressing superficial punctate keratopathy on the ocular surface of patients with this disease. In the current study, we investigated whether rebamipide has anti- inflammatory effects on the ocular surface., Methods: To examine the effects of rebamipide on polyI:C-induced cytokine expression by primary human conjunctival epithelial cells, we used enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction assay. We studied the effects of rebamipide on ocular surface inflammation in our murine experimental allergic conjunctivitis (EAC) model., Results: Rebamipide could suppress polyI:C-induced cytokine production and the expression of mRNAs for CXCL10, CXCL11, RANTES, MCP-1, and IL-6 in human conjunctival epithelial cells. In our EAC model, the topical administration of rebamipide suppressed conjunctival allergic eosinophil infiltration., Conclusions: The topical application of rebamipide on the ocular surface might suppress ocular surface inflammation by suppressing the production of cytokines by ocular surface epithelial cells.

Purpose: To report the effectiveness, disease-specific outcomes, and safety of cultivated oral mucosal epithelial sheet transplantation (COMET), with the primary objective of visual improvement., Design: Noncomparative, retrospective, interventional case series., Participants: This study involved 46 eyes in 40 patients with complete limbal stem cell deficiency (LSCD) who underwent COMET for visual improvement. These LSCD disorders fell into the following 4 categories: Stevens-Johnson syndrome (SJS; 21 eyes), ocular cicatricial pemphigoid (OCP; 10 eyes), thermal or chemical injury (7 eyes), or other diseases (8 eyes)., Methods: Best-corrected visual acuity (BCVA) and ocular surface grading score were examined before surgery; at the 4th, 12th, and 24th postoperative week; and at the last follow-up. Data on COMET-related adverse events and postoperative management were collected. The outcomes in each disease category were evaluated separately., Main Outcome Measures: The primary outcome was the change in median logarithm of the minimum angle of resolution (logMAR) BCVA at the 24th postoperative week. The secondary outcome was the ocular surface grading score., Results: Median logMAR BCVA at baseline was 2.40 (range, 1.10 to 3.00). In SJS, logMAR BCVA improved significantly during the 24 weeks after surgery. In contrast, the BCVA in OCP was improved significantly only at the 4th postoperative week. In 6 of the 7 thermal or chemical injury cases, logMAR BCVA improved after planned penetrating keratoplasty or deep lamellar keratoplasty. Grading scores of ocular surface abnormalities improved in all categories. Of 31 patients with vision loss (logMAR BCVA, >2) at baseline, COMET produced improvement (logMAR BCVA, ≤2) in 15 patients (48%). Visual improvement was maintained with long-term follow-up (median, 28.7 months). Multivariate stepwise logistic regression analysis showed that corneal neovascularization and symblepharon were correlated significantly with logMAR BCVA improvement at the 24th postoperative week (P=0.0023 and P=0.0173, respectively). Although postoperative persistent epithelial defects and slight to moderate corneal infection occurred in the eyes of 16 and 2 patients, respectively, all were treated successfully with no eye perforation., Conclusions: Long-term visual improvement was achievable in cases of complete LSCD. Cultivated oral mucosal epithelial sheet transplantation offered substantial visual improvement even for patients with end-stage severe ocular surface disorders accompanying severe tear deficiency. Patients with corneal blindness such as SJS benefited from critical improvement of visual acuity., Financial Disclosure(s): The author(s) have no proprietary or commercial interest in anymaterials discussed in this article., (Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To assess the surgical combination of autologous cultivated oral mucosal epithelial transplantation and eyelid surgery used to treat patients with severe ocular surface disease and entropion., Design: Observational case series., Methods: Three patients with severe thermal and chemical injury were treated by the surgical combination of autologous cultivated oral mucosal epithelial transplantation and everting sutures to correct entropion. Their clinical outcomes and the efficacy of this surgical procedure were assessed., Results: The ocular surfaces were successfully reconstructed with autologous cultivated oral mucosal epithelial sheets and everting sutures without any complications during the operations. In the course of a mean follow-up period of 30 months their clinical outcomes were assessed. Postoperative follow-up showed that the simultaneous everting sutures caused no problems with the cultivated oral mucosal epithelial sheet, and there were no severe complications such as infection or inflammation. During the follow-up period, in 2 of the 3 eyes the ocular surface and eyelid remained stable with no recurrence of entropion., Conclusion: This case series presents a surgical approach to treat severely scarred ocular surfaces using the combination of autologous cultivated oral mucosal epithelial transplantation and everting sutures. Clinical outcomes suggest that this combined surgical procedure is a safe and useful method for the treatment of patients with severe ocular surface disease and entropion., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: To investigate the long-term outcome of autologous cultivated oral mucosal epithelial transplantation (COMET) for the treatment of the scar phase of severe ocular surface disorders. Participants This study involved 19 eyes of 17 patients who received autologous COMET for total limbal stem-cell deficiency., Methods: Autologous cultivated oral mucosal epithelial sheets were created using amniotic membrane as a substrate. Clinical efficacy was evaluated by best-corrected visual acuity and visual acuity at the postoperative 36th month. The clinical results (clinical conjunctivalisation, corneal opacification, corneal neovascularisation and symblepharon formation) were evaluated and graded on a scale from 0 to 3 according to their severity. Clinical safety was evaluated by the presence of persistent epithelial defects, ocular hypertension and infections., Results: Autologous cultivated oral mucosal epithelial sheets were successfully generated for all 17 patients. All patients were followed up for more than 36 months; the mean follow-up period was 55 months and the longest follow-up period was 90 months. During the long-term follow-up period, postoperative conjunctivalisation and symblepharon were significantly inhibited. All eyes manifested various degrees of postoperative corneal neovascularisation, but it gradually abated and its activity was stable at 6 months after surgery. Best-corrected visual acuity was improved in 18 eyes (95%) during the follow-up periods, and visual acuity at the postoperative 36th month was improved in 10 eyes (53%)., Conclusions: These long-term clinical results strongly support the conclusion that tissue-engineered cultivated oral mucosal epithelial sheets are useful in reconstructing the ocular surface of the scar phase of severe ocular surface disorders.

Background: Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucosa. They often affect the ocular surface and can result in permanent visual dysfunction., Objectives: We sought to discover genetic markers for SJS/TEN susceptibility., Methods: We performed a genome-wide association study with 60 patients and 300 control subjects. We applied stringent filter and visual assessments for selecting single nucleotide polymorphisms (SNPs) and a high false discovery rate threshold. We fine-mapped the region where a candidate SNP was found and confirmed the results by means of sequencing. We evaluated the function of agonist-activated prostaglandin E receptor 3 (EP3), the gene for which contained several SNPs, in regulating cytokine production in human conjunctival epithelial (CE) cells. The expression levels of EP3 in the CE cells from patients and control subjects were also compared., Results: We identified 3 SNPs that passed the false discovery rate threshold. One (rs17131450) was close to the EP3 gene. Therefore we analyzed the EP3 region in detail and identified 5 other SNPs. We confirmed the association between SJS/TEN and all 6 SNPs. Activated EP3 was expressed in control CE cells, and it suppressed polyI:C-stimulated cytokine production, suggesting that EP3 might help prevent ocular surface inflammation. Concordantly, the EP3 levels were much lower in the CE cells of the patients than in those of the control subjects., Conclusion: We demonstrated, using both genetic and functional analyses, that EP3 could be a key player in the pathogenesis of SJS/TEN accompanied by ocular complications., (Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Objective: To evaluate the use of autologous serum (AS)-derived cultivated oral epithelial transplants for the treatment of severe ocular surface disease., Methods: We used AS from 10 patients with severe ocular surface disease and total limbal stem cell deficiency to develop autologous cultivated oral epithelial equivalents. These were compared with epithelial equivalents derived from conventional fetal bovine serum-supplemented medium. Surgery involved removal of the corneal pannus and surrounding diseased tissue and transplantation of the AS-derived epithelial equivalents. The oral equivalents were analyzed by review of histologic and immunohistochemical findings., Results: Oral epithelial sheets cultivated in AS- and fetal bovine serum-supplemented media were similar in morphology, and both formed basement membrane assembly proteins important for maintaining graft integrity. Complete corneal epithelialization was achieved within 2 to 5 days postoperatively. The ocular surface remained stable without major complications in all eyes during a mean +/- SD follow-up of 12.6 +/- 3.9 months. The visual acuity improved by more than 2 lines in 9 of 10 eyes, with transplanted oral epithelium surviving up to 19 months., Conclusion: The successful use of an AS-derived oral epithelial equivalent to treat severe ocular surface disease represents an important advance in the pursuit of completely autologous xenobiotic-free bioengineered ocular equivalents for clinical transplantation.

Purpose: To report an assessment of the two-step surgical combination of cultivated autologous oral mucosal epithelial transplantation (COMET) and penetrating keratoplasty (PKP) used to treat patients with severe limbal deficiency disorders, and to investigate the keratin expression patterns of transplanted surviving oral mucosal epithelium., Design: Observational case series., Methods: Two patients with Stevens-Johnson syndrome and chemical eye injury were treated by COMET followed, approximately six months later, by a PKP triple procedure. In the course of a mean follow-up period of 22.5 months, their clinical outcomes and the efficacy of this two-step surgical procedure were assessed. In addition, the keratin expression in corneal buttons excised during PKP were immunohistochemically examined to characterize the oral mucosal epithelium that survived ectopically on the cornea. In vivo laser confocal microscopy was used to investigate the structure of the epithelium on the corneal grafts., Results: The ocular surfaces were successfully reconstructed with cultivated autologous oral mucosal epithelial sheets and PKP. No clinical complications, such as persistent epithelial defects, rejections, or recurrence of cicatrization, were encountered. Postoperative best-corrected visual acuity was 20/125 in one patient and 20/100 in the other. The surviving oral mucosal epithelium, distinguished by its fluorescence pattern, consisted of an irregular, nonkeratinized, stratified epithelium without goblet cells. Immunohistochemical study demonstrated that K3, but not K12, was expressed in the transplanted cultivated oral mucosal epithelium that was similar to oral mucosal tissue. In vivo, the epithelial structure and cell density in the basal cell layer of the corneal grafts were similar to normal cornea., Conclusions: This study presents a two-step surgical approach to treat severely scarred ocular surfaces by means of a combination of COMET and PKP. Clinical outcomes suggest that this treatment may be beneficial for the maintenance of the reconstructed ocular surface by providing oral mucosal epithelium around the corneal graft.

Purpose: To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS)., Methods: AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry., Results: BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy., Conclusions: AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

Purpose: To perform a midterm assessment of the integrity and reproducibility of cultivated autologous oral mucosal epithelial sheets, and to evaluate the clinical efficacy of their transplantation in ocular surface., Design: Observational case series., Methods: Cultivated autologous oral mucosal epithelial sheets were created using amniotic membrane and buccal mucosal epithelium from 12 patients with Stevens-Johnson syndrome, chemical and thermal injury, pseudo-ocular cicatricial pemphigoid, and idiopathic ocular surface disorder. They were transplanted onto 15 eyes from these patients who were then followed up for a mean of 20 months; with the longest follow-up being 34 months. We assessed their clinical outcomes with special reference to neovascularization., Results: Cultivated autologous oral mucosal epithelial sheets could be generated from all patients. On the second postoperative day, 14 of 15 sheets transplanted demonstrated total re-epithelialization on the cornea. During the follow-up, the ocular surface was stable and transparent without any major complications in 10 of 15 eyes (67%), and the transplanted epithelium survived for at least 34 months. There were five eyes (33%) with small but long-standing epithelial defects, three of these healed spontaneously, and two (13%) required reoperation. In 10 eyes, postoperative visual acuity was improved by more than 2 lines. All eyes manifested some peripheral corneal vascularization., Conclusions: We established a successful tissue-engineering technique to generate cultivated autologous oral mucosal epithelial sheets and succeeded in reconstructing the ocular surface. We suggest that this surgical modality may be both safe and useful, especially in younger patients with the most severe ocular surface disorders.

Background: Patients with severe unilateral ocular surface disease require reconstruction of the damaged ocular surface. We succeeded in culturing primary corneal limbal epithelial cells taken from minimal biopsy and, once grown, transplanting them on denuded amniotic membrane (AM)., Methods: Autologous corneal limbal epithelial cells from a 3 mm(2) biopsy of the uninjured eye were grown for 3 weeks on a denuded AM carrier. The resultant sheet was then transplanted onto the unilateral severely chemically injured eye., Results: Minimal biopsy showed the autologous cultivated corneal epithelial cells to have 4-5 layers of sufficient stratification and to be well differentiated. At 19 months post-transplantation, the ocular surface epithelium was stable and there were no epithelial defects., Conclusion: We document that it is possible to produce sufficiently stratified, well differentiated, autologous cultivated corneal limbal epithelium on AM from a minimal biopsy of the donor eye and to transplant it onto the injured eye.

Purpose: To examine the feasibility of using sterilized, freeze-dried amniotic membrane (FD-AM) as a substrate for cultivating autologous corneal epithelial cells for ocular surface reconstruction., Methods: Human AM deprived of amniotic epithelial cells by incubation with EDTA was freeze dried, vacuum packed, and sterilized with gamma-irradiation. The resultant FD-AM was characterized for its physical, biological, and morphologic properties by stretch stress tests, immunohistochemistry, electron microscopy, and cell culture. In addition, 3 weeks after an ocular surface injury, the conjunctivalized corneal surfaces of eyes in eight rabbits were surgically reconstructed by transplantation of autologous cultivated corneal epithelial cells on FD-AM., Results: A stretch stress test revealed no significant differences between sterilized FD-AM and cryopreserved AM. Immunohistochemistry for several extracellular matrix molecules and electron microscopic analysis of FD-AM revealed that the process of drying and irradiation did not affect its biological and morphologic properties. The corneal epithelial cells cultivated on FD-AM had four to five stratified, well-differentiated cell layers. Corneas that were grafted with the cultivated corneal epithelial cells on FD-AM were clear and were all epithelialized at 10 days after surgery., Conclusions: The sterilized, freeze-dried AM retained most of the physical, biological, and morphologic characteristics of cryopreserved AM; consequently, it is a useful biomaterial for ocular surface reconstruction.

Purpose: Our group performed cultivated allogeneic corneal epithelial transplantation in 13 eyes from 11 patients with severe ocular surface disorders. After the clinical application of this new surgical treatment, some patients experienced epithelial and subepithelial opacities. We applied our procedure again in these patients to achieve successful ocular surface reconstruction., Methods: The corneal limbal epithelial cells from donor corneas were cultivated for 4 weeks on denuded amniotic membrane (AM) carrier, with 3T3 fibroblast coculture and airlifting. The study subjects consisted of 3 patients. At 3 and 12 months after the first operation, the failed epithelial graft with AM was replaced with new allogeneic corneal epithelium cultivated on AM., Results: At 48 hours after transplantation, the corneal surfaces of the 3 eyes were clear and smooth; the entire corneal surfaces were evenly covered with the transplanted cultivated corneal epithelium, which did not stain with fluorescein. The ocular surface epithelia of these patients are all stable without epithelial defects., Conclusions: We have shown that, in cases where the initially transplanted cultivated epithelium becomes opaque, it is possible to repeat the transplantation process with new cultivated epithelium on AM.

Toll-like receptor 3 (TLR3) and interferon-beta promoter stimulator-1 (IPS-1) are associated with antiviral responses to double-stranded RNA viruses and contribute to innate immunity. We previously reported that conjunctival epithelial cell (CEC) TLR3 and IPS-1 pathways respond to the common ligand polyinosinic:polycytidylic acid (polyI:C) to regulate different gene expression patterns as well as CD11c + cell migration in murine-model corneas. However, the differences in the functions and the roles of TLR3 and IPS-1 remain unclear. In this study, we investigated the differences of TLR3 or IPS-1-induced gene expression in corneal epithelial cells (CECs) in response to polyI:C stimulation using cultured murine primary CECs (mPCECs) derived from TLR3 and IPS-1 knockout mice via comprehensive analysis. The genes associated with viral responses were upregulated in the wild-type mice mPCECs after polyI:C stimulation. Among these genes, Neurl3, Irg1, and LIPG were dominantly regulated by TLR3, while interleukin (IL)-6 and IL-15 were dominantly regulated by IPS-1. CCL5, CXCL10, OAS2, Slfn4, TRIM30α, and Gbp9 were complementarily regulated by both TLR3 and IPS-1. Our findings suggest that CECs may contribute to immune responses and that TLR3 and IPS-1 possibly have different functions in the corneal innate immune response. [ABSTRACT FROM AUTHOR]

To evaluate the toxicity of Amphotericin B (AmB) in Optisol™-GS Corneal Storage Media (Bausch & Lomb) on corneal epithelial cell (CEC) morphology and migration ability. Sclerocorneal strips were removed from male Japanese white rabbits, and then stored at 4 °C in Optisol™-GS containing 0 µg/ml of AmB (control group) and 2.5, 5, 25, and 50 µg/ml of AmB (AmB groups; four eyes per group). After 7 days of storage, CEC morphology was evaluated by hematoxylin-eosin staining, immunohistochemical staining (ZO-1), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, to evaluate CEC migration ability, three corneal blocks (6–8 × 3 mm each) from one preserved cornea were cultured for 24 h, and the area of CEC migration (2 mm at the central region) onto the stromal surface was then measured. At 5, 25, and 50 µg/ml of AmB, deformation and vacuolation of CECs were observed in all preserved corneas. ZO-1 expression was significantly reduced in corneas preserved at AmB concentrations of 25 and 50 µg/ml. TUNEL Labeling Index was significantly increased at AmB concentrations of ≥5 µg/ml. CEC migration was inhibited in a dose-dependent manner at AmB concentrations of 25 and 50 µg/ml compared to the control group. The addition of AmB to Optisol™-GS can be toxic to CECs and inhibit their migration at a concentration of ≥5 µg/ml. AmB at a concentration of 2.5 µg/ml can be considered safe for the preservation of donor corneal tissue used in corneal epithelial transplantation surgery. [ABSTRACT FROM AUTHOR]

Nasolacrimal duct obstruction (NLDO) is thought to be due to inflammation and fibrosis of lacrimal duct epithelial cells (LDECs). Here we investigated the effect of rebamipide, a drug that is used for the protection of the mucosa and the treatment of gastritis and gastroduodenal ulcers, on LDECs, both in vitro and in vivo. In this study, LDECs were cultured from rabbit lacrimal duct tissues, and the barrier function of LEDCs was examined in vitro via transepithelial electrical resistance (TER) measurement, with or without interleukin (IL)-6 and/or rebamipide. For the in vivo examination, benzalkonium chloride (BAC) was injected into the rabbit lacrimal ducts, followed by the application of rebamipide or a placebo vehicle alone. The results of the in vitro examination revealed a significant decrease in TER in the group treated with IL-6 alone compared with the placebo-vehicle group (p < 0.05) and the group treated with IL-6 and rebamipide (p < 0.01). The results of the in vivo examination revealed that the infiltration of neutrophils under the basement membrane and the disruption of tight junction proteins with BAC injection and rebamipide attenuates the disturbance of tissue construction. These results suggest that rebamipide protects LDECs via an anti-inflammatory effect and preserves the barrier function of those cells. [ABSTRACT FROM AUTHOR]

Purpose: We previously reported that human conjunctival epithelial cells expressed functioning interleukin-4 receptor α (IL-4Rα). In this study, we investigated whether human corneal epithelial cells also express functioning IL-4Rα. Methods: The presence of IL-4Rα mRNA and protein in human corneal epithelium was examined by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistology, respectively. The cell surface expression of IL-4Rα and the transcripts upregulated upon IL-4Rα ligand (IL-4 or IL-13) stimulation were examined by flow cytometry and quantitative RT-PCR, respectively, using immortalized human corneal-limbal epithelial (HCLE) cells. Results: The mRNA and protein of IL-4Rα were detected in human corneal epithelium. Flow cytometry analysis showed the cell surface expression of IL-4Rα protein. Quantitative RT-PCR assay of HCLE cells showed the upregulation of the transcripts tumor necrosis factor alpha-induced protein 6 (TNFAIP6), RAS guanyl-releasing protein 1 (RASGRP1), carbonic anhydrase II (CA2), cytokine-inducible SH2-containing protein (CISH), hyaluronan synthase 3 (HAS3), calpain 14 (CAPN14), endothelin receptor type A (EDNRA), cathepsin C (CTSC), and lecithin retinol acyltransferase (LRAT) as well as human conjunctival epithelial cells. Conclusion: Human corneal epithelial cells expressed functioning IL-4Rα, and stimulation of its ligands, IL-4 and IL-13, could induce the expression of various genes, e.g., antiinflammatory molecule genes such as TNFAIP6 and CISH and cellular differentiation and proliferation-related molecule genes such as RASGRP1, HAS3, EDNRA, and LRAT. [ABSTRACT FROM AUTHOR]

PURPOSE. Previously we reported the occurrence of estrogen receptor α (ERβ), estrogen receptor β (ERβ) and androgen receptor (AR) in mouse corneas. The present study was designed to investigate the occurrence of various sex steroid hormone receptors, including ERα, progesterone receptor (PR) and AR, in human corneas. METHODS. We used reverse transcription-polymerase chain reaction (RT-PCR) to look for sex hormone receptor mRNAs (ERα, PR and AR) in human corneal epithelial cells obtained at autopsy. Next, using an immunocytochemical technique, we localized these receptors in donor human corneas. RESULTS. mRNAs encoding all receptors tested for were found in corneal epithelial cells obtained from male and female donor eyes. Immunocytochemical examination revealed that the receptors were located in the nuclei of corneal epithelial, stromal, and endothelial cells. CONCLUSIONS. Since receptors for both male and female sex hormones are present in human corneas of both genders, we postulate that the receptors may influence the biological functions of corneal cells through direct interaction with specific hormones. [ABSTRACT FROM AUTHOR]

PURPOSE. The topical administration of 1α,25-dihydroxy-vitamin D3 [1α,25(OH)2D3 ] inhibits Langerhans cell (LC) migration and corneal neovascularization in mice. Since the cytokines that induce LC migration [e.g. interleukin-1 (IL-1)] and corneal neovascularization [e.g. interleukin-8 (IL-8)] are produced by human corneal epithelial cells, we investigated the inhibitory effects of 1α,25(OH)2D 3 on cytokine production by these cells in vitro. METHODS. In this experiment, human corneal epithelial cells, cultured in DMEM-FBS until confluence, were then switched to serum-free DMEM containing insulin, transferrin, and sodium selenite (DMEM-ITS) for 48 hours. Next, they were cultured with DMEM-ITS containing 1α,25(OH)2D3 at concentrations of 10-7 M, 10-11 M, or 10 –15 M, and vehicle only (0.1% ethanol). After 6 or 12 hours in this culture, the supernatants were collected and concentrations of IL-1α, IL-1b, and IL-8 were quantified by ELISA. RESULTS. Significantly lower levels of IL-1α and IL-1b were detected in supernatants from cells cultured with 1α,25(OH)2D 3 (10-7 M, 10-11 M, and 10-15 M), compared to cells cultured with vehicle only. This was true at 6 and 12 hours after the addition of 1α,25(OH)2D3 (p < 0.05). IL-8 production inhibition by 1α,25(OH)2D3, on the other hand, was detected at 6 hours (p < 0.0005) but not at 12 hours (p > 0.1). CONCLUSIONS. 1α,25(OH)2D3 inhibits cytokine (IL-1α, IL-1b, and IL-8) production by human corneal epithelial cells in vitro. We suspect that 1α,25(OH)2D3 can inhibit LC migration and corneal neovascularization, as is seen in ocular surface inflammation. [ABSTRACT FROM AUTHOR]

Purpose: To determine the long-term epithelial lineage of origin of surgically removed grafts after allogeneic cultivated corneal limbal epithelial transplantation (CLET). Design: Interventional case reports. Participants: We studied 2 eyes from 2 patients with total corneal stem cell destruction; 1 eye was from a patient with Stevens-Johnson syndrome and 1 eye had sustained chemical injury. Methods: Allogeneic cultivated corneal limbal epithelial sheets on human amniotic membrane (AM) were transplanted onto the ocular surface. Regrafting (1 eye, 42 months later) or penetrating keratoplasty (1 eye, 75 months later) were performed after the initial transplantation procedure for further visual rehabilitation. Main Outcome Measures: The excised grafts were subjected to clinical evaluation and to light- and transmission electron microscopy (TEM) examination and to immunohistochemical analysis. Results: In clinically conjunctival grafts, TEM and immunohistochemical analysis disclosed only small areas where the original cultivated corneal epithelial cells persisted. Neighboring conjunctival epithelial cells had apparently invaded a large portion of the corneal surface (keratin 3/12(−), Muc5ac(+)). In clinically corneal grafts, transplanted allogeneic cultivated corneal epithelial cells clearly survived for a long period of time (keratin 3/12(+), Muc5ac(−)); there was no infiltration by inflammatory cells, nor was there dissolution of the AM substrate. Conclusions: We theorize that the process of graft opacification after allogeneic CLET is responsible for the loss of transplanted cultivated corneal epithelial cells and that this is followed by conjunctival cell invasion onto the corneal surface. The results of this study confirmed that in the clinically evaluated corneal graft, transplanted cultivated corneal epithelial cells indeed survived for a long period of time on the corneal surface and maintained ocular surface integrity, even though the transplanted cells were allogeneic. Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article. [Copyright &y& Elsevier]

To clarify the influence of tumor necrosis factor (TNF)-α on fibrotic phenotypes induced by transforming growth factor (TGF)-β in retinal pigment epithelial cells (RPECs) by epigenetic regulation. Human primary retinal pigment epithelial cells (RPECs including ARPE19) were used in cultures in the presence or absence of TNF-α and/or TGF-β2. RT2 Profiler™ (Qiagen) was used for PCR Array for fibrosis and epithelial mesenchymal transition (EMT). Microarray analysis by 3D gene DNA chip was outsourced to Toray Industries Inc. Quantification of histone acetyl transferase (HAT)-related and histone deacetylase (HDAC) related gene expression were also analyzed. HDAC and HAT activity was measured using an EpiQuik HDAC and HAT Activity/Inhibition Assay Kit (Epigentek). CD44, MMP-9, HAT, and HDAC in RPECs were analyzed by western blotting. Analysis of expression of the EMT/fibrosis related CD44 and MMP-9 phenotypes induced by TNF-α+TGF-β2 revealed four alterations in RPECs: 1) abolition of TGF-β2-induced α-SMA by TNF-α; 2) synergy between TNF-α+TGF-β2 for induction of CD44 and MMP-9 phenotypes 3) no inhibition of HDAC activity by either TNF-α or TGF-β2; and 4) significant inhibition of HAT activity by either TNF-α or TGF-β2, but no synergy. The HDAC activation through HAT inhibition by TNF-α+TGF-β was counteracted by HDAC inhibitors, leading to the inhibition of EMT/fibrosis. EMT/fibrotic CD44 and MMP-9 phenotypes were epigenetically upregulated by concerted action of TNF-α and TGF-β in RPECs. The intervention in epigenetic regulation may hold potential in preventing intraocular proliferative diseases. • TNF-α+TGF-β2 epigenetically upregulated CD44 and MMP-9 phenotypes in RPECs. • HAT activity was inhibited by either TNF-α or TGF-β2, but no synergy. • HAT inhibition by TNF-α+TGF-β was counteracted by HDAC inhibitors. [ABSTRACT FROM AUTHOR]

A healthy corneal epithelium, which is essential for proper vision and protection from external pathogens, is continuously replenished throughout life by stem cells located at the limbus. In diseased or injured eyes, however, in which stem cells are deficient, severe ocular problems manifest themselves. These are notoriously difficult to manage and as a result the last 20 or so years has seen a number of therapeutic strategies emerge that aim to recover the ocular surface and restore vision in limbal stem cell deficient eyes. The dominant concept involves the generation of laboratory cultivated epithelial cell sheets expanded from small biopsies of the epithelial limbus (for patient or donors) or another non-corneal epithelial tissue such as the oral mucosa. Typically, cells are grown on sterilised human amniotic membrane as a substrate, which then forms part of the graft, or specially formulated plastic culture dishes from which cells sheets can be released by lowering the temperature, and thus the adherence of the plastic to the cells. Overall, clinical results are promising, as is discussed, with new cultivation methodologies and different cell lineages currently being investigated to augment the treatment options for visual disturbance caused by a corneal epithelial limbal stem cell deficiency. [ABSTRACT FROM AUTHOR]

PAX6, a paired box transcription factor, is necessary for eye development. However, how it regulates the cell identity of human corneal epithelial cells (CECs) is not well understood. We aimed to clarify the function of PAX6 in human CECs using gene knockout via the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system. We designed guide RNAs for different targets in PAX6. PAX6-depleted CECs maintained the epithelial morphology, but became larger. Global analyses using microarray revealed that down-regulated genes were primarily CEC-specific and included keratin 12, keratin 3, clusterin (CLU), aldehyde dehydrogenase 3 family member A1 (ALDH3A1), angiopoietin-like 7 (ANGPTL7) and transketolase (TKT), while up-regulated genes were primarily epidermis-related and included keratin 10, keratin 1, involucrin (IVL), filaggrin (FLG). These findings suggest that PAX6 maintains CEC identity by regulating differentiation. [ABSTRACT FROM AUTHOR]

The aim of the study is to clarify the participation of extracellular vesicles (EV) secreted by murine primary retinal pigment epithelial (mpRPE) cells in the cell to cell communication with macrophages (Mps), firstly described by the authors in 2016. In ocular inflammation, Mps act as sources of tumor necrosis factor-α (TNF-α), an activator of RPE cells. TNF-α stimulates the production of monocyte chemotactic protein (MCP-1) by RPE cells, thereby causing greater recruitment of Mps to the sub-RPE space. Murine RAW 264.7 Mps cells were co-cultured with C57BL/6 mouse mpRPE cells, either together or separated in transwells, vertically or horizontally connectable, with 0.40 or 0.03 μm membrane filters. The association of EV with mpRPE or RAW 264.7 was quantified by fluorescence cell sorting (FACS) using Qdot655 streptavidin-conjugated biotinylated EV. Increased levels of CD63+ EV were detected in co-cultures by western blotting or FACS analysis, in accordance with the increased production of nanoparticles (50–150 nm) detected by Nanosight tracking analysis. The gene expressions of cytokines, MCP-1, IL-6, IL-8, and VEGF in mpRPE cells and the corresponding proteins were increased in co-cultures even in transwells, vertically connected with 0.40 μm membrane filters, while the repressed TNF-α protein production was not affected. Most of the CD63+ EVs produced by mpRPE cells in co-cultures were associated with Raw264.7, but not with mpRPE cells. Semi-purified CD63+ EV secreted from mpRPE cells, increased the secretion of MCP-1, IL-6, and VEGF in co-cultures with RAW 264.7. Culture chamber separation horizontally connected with 0.03 μm membrane filters reduced this increased secretion. Collectively, mpRPE derived CD63+ EV partly participate in the sub-retinal innate inflammation. To evaluate the functional damage of RPE cells upon chronic exposure to here defined EVs will be the critical issue to uncover their roles in chronic retinal degenerative diseases. • Secretion of CD63+ extracellular vesicles (EVs) was increased in co-cultures of mouse primary (mp) RPE cells with RAW264.7. • The increase of cytokines MCP-1, IL-6, IL-8, and VEGF was prevented when co-cultured in chambers with a 0.03 μm filter. • CD63+ EVs produced by mpRPE cells may participate in sub-retinal inflammation by inducing TNF-α expression in macrophages. [ABSTRACT FROM AUTHOR]