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Langer, Marian G., C. V. Sundarraj e Nirmala Sundarraj. "Corneal epithelial-specific cell surface antigen recognized by a monoclonal antibody". Development 94, n.º 1 (1 de junho de 1986): 163–72. http://dx.doi.org/10.1242/dev.94.1.163.
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Monoclonal antibodies, specific against cell surface differentiation antigens of human corneal epithelial cells, were developed using epithelial cells resected from human corneas as the immunogens. One of these antibodies reacted specifically with corneal epithelial cells and not with epithelial cells of other tissues when tested by an indirect immunoperoxidase technique. Nonidet P-40 extracts of different subcellular fractions of human corneal epithelial cells were tested for their reactivity against this antibody using an enzyme-linked immunosorbent assay. The results indicated that the antigen recognized by this antibody is associated with the plasma membrane. This was further verified by immuno-electron-microscopic analysis using ferritin-conjugated anti-mouse IgG antibody. This antigen was not detectable in the corneal epithelial cells in primary cultures nor in the epithelial cells from early stages of developing cornea (12 to 18 weeks in utero) but was present in the epithelial cells in the corneas of an 8-month-old infant. Therefore, this surface-associated antigen identified in the present study is a developmentally regulated marker of human corneal epithelium.
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Alarcon, Irania, Lesley Kwan, Chong Yu, David J. Evans e Suzanne M. J. Fleiszig. "Role of the Corneal Epithelial Basement Membrane in Ocular Defense against Pseudomonas aeruginosa". Infection and Immunity 77, n.º 8 (8 de junho de 2009): 3264–71. http://dx.doi.org/10.1128/iai.00111-09.
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ABSTRACT Pseudomonas aeruginosa can invade corneal epithelial cells and translocates multilayered corneal epithelia in vitro, but it does not penetrate the intact corneal epithelium in vivo. In healthy corneas, the epithelium is separated from the underlying stroma by a basement membrane containing extracellular matrix proteins and pores smaller than bacteria. Here we used in vivo and in vitro models to investigate the potential of the basement membrane to defend against P. aeruginosa. Transmission electron microscopy of infected mouse corneas in vivo showed penetration of the stroma by P. aeruginosa only where the basement membrane was visibly disrupted by scratch injury, suggesting that the intact basement membrane prevented penetration. This hypothesis was explored using an in vitro Matrigel Transwell model to mimic the corneal basement membrane. P. aeruginosa translocation of multilayered corneal epithelia grown on Matrigel was ∼100-fold lower than that of cells grown without Matrigel (P < 0.005, t test). Matrigel did not increase transepithelial resistance. Matrigel-grown cells blocked translocation by a P. aeruginosa protease mutant. Without cells, Matrigel also reduced traversal of P. aeruginosa and the protease mutant. Fluorescence microscopy revealed a relative accumulation of bacteria at the superficial epithelium of cells grown on Matrigel at 3 h compared to cells grown on uncoated filters. By 5 h, bacteria accumulated beneath the cells, suggesting direct trapping by the Matrigel. These findings suggest that the basement membrane helps defend the cornea against infection via physical barrier effects and influences on the epithelium and that these roles could be compromised by P. aeruginosa proteases.
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Hazlett, L. D., e P. Mathieu. "Glycoconjugates on corneal epithelial surface: effect of neuraminidase treatment." Journal of Histochemistry & Cytochemistry 37, n.º 8 (agosto de 1989): 1215–24. http://dx.doi.org/10.1177/37.8.2754252.
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The purpose of this study was to develop a procedure to quantitatively examine corneal epithelial apical cell membrane-associated glycoconjugates. Saccharide moieties on young, mature, and aged corneal epithelial cells were detected and localized in corneas of immature and adult mice by using colloidal gold-labeled lectins and transmission electron microscopy (TEM). In general, dense binding to the corneal epithelial apical surface cell membranes with wheat germ agglutinin (WGA) was seen in the adult, whereas the immature cornea bound less WGA-gold. Neuraminidase digestion decreased binding of the conjugate on epithelial plasma membranes of young and mature cells in adult cornea. Lectin-gold binding was decreased in the immature cornea on mature and aged cells. WGA-gold binding after neuraminidase was elevated on young cells of immature and on aged cells of adult animals. No binding of peanut agglutinin (PNA) or horse gram agglutinin (DBA) to the corneal epithelial surface was seen in animals of either age. After neuraminidase digestion, PNA binding sites were exposed only on the adult corneal surface. These data suggest that a terminal trisaccharide sequence, sialic acid-galactose beta(1----3)-N-acetylgalactosamine, is present at the adult corneal surface but is absent or at undetectable levels at the corneal surface of the immature animal. These data may be of significance in light of the dissimilar pattern of P. aeruginosa recognition and binding to the immature vs adult corneal epithelium.
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Augustin, Danielle K., Susan R. Heimer, Connie Tam, Wing Y. Li, Jeff M. Le Due, David J. Evans e Suzanne M. J. Fleiszig. "Role of Defensins in Corneal Epithelial Barrier Function againstPseudomonas aeruginosaTraversal". Infection and Immunity 79, n.º 2 (29 de novembro de 2010): 595–605. http://dx.doi.org/10.1128/iai.00854-10.
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ABSTRACTStudies have shown that epithelium-expressed antimicrobial peptides (AMPs), e.g., β-defensins, play a role in clearing bacteria from mouse corneas already infected withPseudomonas aeruginosa. Less is known about the role of AMPs in allowing the cornea to resist infection when healthy. We previously reported that contact lens exposure, a major cause ofP. aeruginosakeratitis, can inhibit the upregulation of human β-defensin 2 (hBD-2) by corneal epithelial cells in response toP. aeruginosaantigensin vitro. Here, we studied the role of AMPs in maintaining the corneal epithelial barrier toP. aeruginosapenetration using bothin vitro(human) andin vivo(mouse) experiments. Results showed that preexposing human corneal epithelial multilayers to bacterial antigens in a culture supernatant (known to upregulate AMP expression) reduced epithelial susceptibility toP. aeruginosatraversal up to 6-fold (P< 0.001). Accordingly, small interfering RNA (siRNA) knockdown of any one of four AMPs expressed by human epithelia promotedP. aeruginosatraversal by more than 3-fold (P< 0.001). The combination knockdown of AMPs further enhanced susceptibility to bacterial traversal by ∼8-fold (P< 0.001).In vivoexperiments showed that the loss of murine β-defensin 3 (mBD-3), a murine ortholog of hBD-2, enhanced corneal susceptibility toP. aeruginosa. The uninjured ocular surface of mBD-3−/−mice showed a reduced capacity to clearP. aeruginosa, and their corneal epithelia were more susceptible to bacterial colonization, even when inoculatedex vivoto exclude tear fluid effects. Together, thesein vitroandin vivodata show functional roles for AMPs in normal corneal epithelial cell barrier function againstP. aeruginosa.
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Zieske, J. D., e M. Wasson. "Regional variation in distribution of EGF receptor in developing and adult corneal epithelium". Journal of Cell Science 106, n.º 1 (1 de setembro de 1993): 145–52. http://dx.doi.org/10.1242/jcs.106.1.145.
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Epidermal growth factor receptor has been localized to the proliferative cell layers in a variety of stratified squamous epithelia. In the current study, the rat cornea was used as an experimental model to determine if epidermal growth factor receptor is concentrated in epithelial stem cells. Epidermal growth factor receptor was localized using immunofluorescence microscopy in adult and neonatal (1-day to 4-week) rat corneas. Antibody binding to epidermal growth factor receptor was present in basal cells across the adult cornea but was more intense in the limbal zone. In rats 1 day to 1 week of age, the corneal epithelium consisted of one or two layer of cells that were intensely labeled by anti-epidermal growth factor receptor. Following epithelial stratification, which occurred just prior to eyelid opening (approximately 12 days), expression of epidermal growth factor receptor was greatly reduced in central corneal epithelium and gained an adult pattern by 3 weeks of age. Expression of epidermal growth factor receptor was also examined by incubating 1 mm slices of adult corneas with 125I-epidermal growth factor (4 nM) for 90 minutes, followed by washing and autoradiography. Basal cells in the limbal zone contained 4.5-fold more silver grains per cell than did basal cells in the central cornea. These data suggest that cells with high potential for proliferation, i.e. limbal basal cells and all basal cells in developing rats, express high epidermal growth factor receptor levels. High levels of receptor may allow these cells to be rapidly stimulated by growth factors to undergo cell division during development and following wounding in adult corneas.(ABSTRACT TRUNCATED AT 250 WORDS)
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Park, Mijeong, Alexander Richardson, Elvis Pandzic, Erwin P. Lobo, J. Guy Lyons e Nick Di Girolamo. "Peripheral (not central) corneal epithelia contribute to the closure of an annular debridement injury". Proceedings of the National Academy of Sciences 116, n.º 52 (16 de dezembro de 2019): 26633–43. http://dx.doi.org/10.1073/pnas.1912260116.
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Corneal epithelia have limited self-renewal and therefore reparative capacity. They are continuously replaced by transient amplifying cells which spawn from stem cells and migrate from the periphery. Because this view has recently been challenged, our goal was to resolve the conflict by giving mice annular injuries in different locations within the corneolimbal epithelium, then spatiotemporally fate-mapping cell behavior during healing. Under these conditions, elevated proliferation was observed in the periphery but not the center, and wounds predominantly resolved by centripetally migrating limbal epithelia. After wound closure, the central corneal epithelium was completely replaced by K14+limbal-derived clones, an observation supported by high-resolution fluorescence imaging of genetically marked cells in organ-cultured corneas and via computational modeling. These results solidify the essential role of K14+limbal epithelial stem cells for wound healing and refute the notion that stem cells exist within the central cornea and that their progeny have the capacity to migrate centrifugally.
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Han, Sang Beom, Farah Nur Ilyana Mohd Ibrahim, Yu-Chi Liu e Jodhbir S. Mehta. "Efficacy of Modified Amnion-Assisted Conjunctival Epithelial Redirection (ACER) for Partial Limbal Stem Cell Deficiency". Medicina 57, n.º 4 (10 de abril de 2021): 369. http://dx.doi.org/10.3390/medicina57040369.
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Background and objectives: the aim of this study was to analyze the efficacy of a modified “amnion-assisted conjunctival epithelial redirection (ACER)” technique for the treatment of partial limbal stem cell deficiency (LSCD). Materials and methods: the medical records of three patients with partial LSCD who underwent corneal surface reconstruction with modified ACER following superficial keratectomy were retrospectively studied. Briefly, in this technique, an inner amniotic membrane (AM) layer was applied on the corneal surface to promote corneal re-epithelialization. The outer AM layer was applied as a barrier to prevent the invasion of conjunctival epithelial cells into the cornea before the corneal surface was completely covered by corneal epithelial cells derived from the remaining intact limbal stem cells. Results: in all three cases, the outer AM layer successfully kept the conjunctival epithelium away from the corneal surface and prevented an admixture of conjunctival epithelial cells with corneal epithelial cells. In all three patients, the cornea was completely re-epithelized with epithelial cells derived from the remaining healthy limbal stem cells, and a clear visual axis was maintained without recurrence for a mean follow-up period of 37.3 ± 8.6 months. Conclusions: the preliminary results suggest that modified ACER appears to be a viable option for patients with partial LSCD.
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Ryu, Jin Suk, So Yeon Kim, Mee Kum Kim e Joo Youn Oh. "Inflammation Confers Healing Advantage to Corneal Epithelium following Subsequent Injury". International Journal of Molecular Sciences 24, n.º 4 (7 de fevereiro de 2023): 3329. http://dx.doi.org/10.3390/ijms24043329.
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Recent evidence shows that epithelial stem/progenitor cells in barrier tissues such as the skin, airways and intestines retain a memory of previous injuries, which enables tissues to accelerate barrier restoration after subsequent injuries. The corneal epithelium, the outermost layer of the cornea, is the frontline barrier for the eye and is maintained by epithelial stem/progenitor cells in the limbus. Herein, we provide evidence that inflammatory memory also exists in the cornea. In mice, eyes that had been exposed to corneal epithelial injury exhibited faster re-epithelialization of the cornea and lower levels of inflammatory cytokines following subsequent injury (either the same or a different type of injury) relative to naïve eyes without previous injury. In ocular Sjögren’s syndrome patients, corneal punctate epithelial erosions were significantly reduced after experiencing infectious injury compared with before. These results demonstrate that previous exposure of the corneal epithelium to inflammatory stimuli enhances corneal wound healing in response to a secondary assault, a phenomenon which points to the presence of nonspecific inflammatory memory in the cornea.
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Schermer, A., S. Galvin e T. T. Sun. "Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells." Journal of Cell Biology 103, n.º 1 (1 de julho de 1986): 49–62. http://dx.doi.org/10.1083/jcb.103.1.49.
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In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."
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Bardag-Gorce, Fawzia, Alissa Diaz, Robert Niihara, Jeremy Stark, Daileen Cortez, Alexander Lee, Richard Hoft e Yutaka Niihara. "Aldehyde Dehydrogenases Expression in Corneal Epithelial Cells with Limbal Stem Cell Deficiency". International Journal of Molecular Sciences 23, n.º 7 (5 de abril de 2022): 4032. http://dx.doi.org/10.3390/ijms23074032.
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Purpose: The purpose of the present study is to investigate the expression of aldehyde dehydrogenases (ALDHs) in rabbit corneas with limbal stem cell deficiency (LSCD) and corneas treated with cultured autologous oral mucosa epithelial cell sheet CAOMECS designed to reconstruct the ocular surface with LSCD. Methods: New Zealand white rabbit autologous oral mucosal epithelial cells were isolated from a buccal biopsy and cultured to be grafted back onto corneas of rabbit model of LSCD. Immunofluorescent staining and Western blot analysis were used to compare the expression of ALDH1A1 and ALDH1A3 in healthy, LSCD-diseased, CAOMECS treated corneas. Human oral mucosal and corneal epithelial cells (OMECS and CECs) were cultured and treated with retinoic acid (RA) to further investigate the expression of ALDHs. Results: In healthy corneas, ALDH1A1 and ALDH1A3 were markedly expressed in basal cells of corneal epithelium. In LSCD diseased corneas, ALDH1A1 and ALDH1A3 were markedly expressed in the conjunctivalized apical epithelial cells, the goblet cells, and the stroma. CAOMECS grafted corneas showed a decreased expression of ALDHs as compared to LSCD diseased corneas. Western blot analysis confirmed the up regulation of ALDH1A1 and ALDH1A3 expression in LSCD-diseased corneal epithelial cells. CAOMECS expressed low levels of ALDH1A1 and ALDH1A3, as compared to diseased CECs (D-CEC). When ALDH1A3 was up regulated by retinoic acid treatment in OMECS, Pax-6 expression was down regulated, suggesting a decrease in regenerative capacity when ALDH enzymes are up regulated. Conclusions: These findings report for the first time the up regulation of ALDH1A1 and ALDH1A3 in rabbit corneas with LSCD and document that CAOMECS grafting used to reconstruct corneal epithelium may reduce the expression levels of ALDH enzymes.
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Findlay, Amy S., D. Alessio Panzica, Petr Walczysko, Amy B. Holt, Deborah J. Henderson, John D. West, Ann M. Rajnicek e J. Martin Collinson. "The core planar cell polarity gene, Vangl2 , directs adult corneal epithelial cell alignment and migration". Royal Society Open Science 3, n.º 10 (outubro de 2016): 160658. http://dx.doi.org/10.1098/rsos.160658.
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This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro . Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration.
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Pino, Christopher J., Frederick R. Haselton e Min S. Chang. "Seeding of Corneal Wounds by Epithelial Cell Transfer from Micropatterned PDMS Contact Lenses". Cell Transplantation 14, n.º 8 (setembro de 2005): 565–71. http://dx.doi.org/10.3727/000000005783982783.
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Persistent corneal wounds result from numerous eye disorders, and to date, available treatments often fail to accelerate reepithelialization, the key initial step in wound healing. To speed reepithelialization, we explored a cell-transfer transplant method utilizing polydimethylsiloxane (PDMS) contact lenses to deliver epithelial cells derived from limbal explants directly within a corneal wound. Human primary epithelial cells and an immortalized corneal epithelial cell line (HCE-SV40) grew well on PDMS contact lenses and their morphology and growth rates where similar to cells grown on tissue culture polystyrene. To initially study cell transfer from PDMS, HCE-SV40 cells were seeded onto PDMS with or without micropatterned posts. After a day in culture, HCE-SV40 cells attached to the unpatterned PDMS uniformly, whereas on micropatterned PDMS they appeared to attach primarily between posts. The cell-covered PDMS contacts were then placed cell-side down onto tissue culture plastic and, after 1, 2, or 3 days, the PDMS contact was removed and the transferred cells were trypsinized and counted. Micropatterned PDMS contact lenses with 100-μm-diameter posts and a post height of 40 μm transferred three times as many cells as unpatterned PDMS. Cell transfer to a wounded cornea was tested in a pig cornea organ culture model deepithelialized by alkali treatment. Post micropatterned PDMS contact lenses were seeded with labeled HCE-SV40 cells at a density 50,000 cells/cm2 and applied to the wounded pig corneas. After 24, 48, or 96 h of application, PDMS contact lenses were removed, corneas fixed with formaldehyde, and sectioned. After 48 h, epithelial cells transferred from post micropatterned contact lenses to provide 35% epithelial coverage of denuded pig corneas; after 96 h coverage was 65%. We conclude that cell transfer from epithelial-coated PDMS contact lenses micropatterned with posts provides a promising approach to reepithelialize corneal surfaces.
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Becker, Ulrich, Carsten Ehrhardt, Marc Schneider, Leon Muys, Dorothea Gross, Klaus Eschmann, Ulrich F. Schaefer e Claus-Michael Lehr. "A Comparative Evaluation of Corneal Epithelial Cell Cultures for Assessing Ocular Permeability". Alternatives to Laboratory Animals 36, n.º 1 (fevereiro de 2008): 33–44. http://dx.doi.org/10.1177/026119290803600106.
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The purpose of this study was to evaluate the potential value of different epithelial cell culture systems as in vitro models for studying corneal permeability. Transformed human corneal epithelial (HCE-T) cells and Statens Serum Institut rabbit corneal (SIRC) cells were cultured on permeable filters. SkinEthic human corneal epithelium (S-HCE) and Clonetics human corneal epithelium (C-HCE) were received as ready-to-use systems. Excised rabbit corneas (ERCs) and human corneas (EHCs) were mounted in Ussing chambers, and used as references. Barrier properties were assessed by measuring transepithelial electrical resistance, and by determining the apparent permeability of markers with different physico–chemical properties, namely, fluorescein, sodium salt; propranolol hydrochloride; moxaverine hydrochloride; timolol hydrogenmaleate; and rhodamine 123. SIRC cells and the S-HCE failed to develop epithelial barrier properties, and hence were unable to distinguish between the permeation markers. Barrier function and the power to differentiate compound permeabilities were evident with HCE-T cells, and were even more pronounced in the case of C-HCE, corresponding very well with data from ERCs and EHCs. A net secretion of rhodamine 123 was not observed with any of the models, suggesting that P-glycoprotein or similar efflux systems have no significant effects on corneal permeability. Currently available corneal epithelial cell culture systems show differences in epithelial barrier function. Systems lacking functional cell–cell contacts are of limited value for assessing corneal permeability, and should be critically evaluated for other purposes.
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Musayeva, Aytan, Caroline Manicam, Andreas Steege, Christoph Brochhausen, Beate K. Straub, Katharina Bell, Norbert Pfeiffer e Adrian Gericke. "Role of α1-adrenoceptor subtypes on corneal epithelial thickness and cell proliferation in mice". American Journal of Physiology-Cell Physiology 315, n.º 5 (1 de novembro de 2018): C757—C765. http://dx.doi.org/10.1152/ajpcell.00314.2018.
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Adrenergic stimuli are important for corneal epithelial structure and healing. The purpose of the present study was to examine the hypothesis that the lack of a single α1-adrenoceptor (α1-AR) subtype affects corneal epithelial thickness and cell proliferation. Expression levels of α1-AR mRNA were determined in mouse cornea using real-time PCR. In mice devoid of one of the three α1-AR subtypes (α1A-AR−/−, α1B-AR−/−, α1D-AR−/−) and in wild-type controls, thickness of individual corneal layers, the number of epithelial cell layers, and average epithelial cell size were determined in cryosections. Endothelial cell density and morphology were calculated in corneal explants, and epithelial cell proliferation rate was determined with immunofluorescence microscopy. Moreover, the ultrastructure of the corneal epithelium was examined by transmission electron microscopy. Messenger RNA for all three α1-AR subtypes was expressed in whole cornea and in corneal epithelium from wild-type mice with a rank order of abundance of α1A ≥ α1B > α1D. In contrast, no α1-AR mRNA was detected in the stroma, and only α1B-AR mRNA was found in the Descemet endothelial complex. Remarkably, corneal epithelial thickness and mean epithelial cell size were reduced in α1A-AR−/− mice. Our findings suggest that the α1A-AR exerts growth effects in mouse corneal epithelial cells.
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Nuzzi, Alessia, Francesco Pozzo Giuffrida, Saverio Luccarelli e Paolo Nucci. "Corneal Epithelial Regeneration: Old and New Perspectives". International Journal of Molecular Sciences 23, n.º 21 (28 de outubro de 2022): 13114. http://dx.doi.org/10.3390/ijms232113114.
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Corneal blindness is the fifth leading cause of blindness worldwide, and therapeutic options are still often limited to corneal transplantation. The corneal epithelium has a strong barrier function, and regeneration is highly dependent on limbal stem cell proliferation and basement membrane remodeling. As a result of the lack of corneal donor tissues, regenerative medicine for corneal diseases affecting the epithelium is an area with quite advanced basic and clinical research. Surgery still plays a prominent role in the treatment of epithelial diseases; indeed, innovative surgical techniques have been developed to transplant corneal and non-corneal stem cells onto diseased corneas for epithelial regeneration applications. The main goal of applying regenerative medicine to clinical practice is to restore function by providing viable cells based on the use of a novel therapeutic approach to generate biological substitutes and improve tissue functions. Interest in corneal epithelium rehabilitation medicine is rapidly growing, given the exposure of the corneal outer layers to external insults. Here, we performed a review of basic, clinical and surgical research reports on regenerative medicine for corneal epithelial disorders, classifying therapeutic approaches according to their macro- or microscopic target, i.e., into cellular or subcellular therapies, respectively.
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Wang, Fan, Bo Ren e Yi Ning Yan. "Stem Cell-Like Characteristics of Corneal Epithelial Cells". Advanced Materials Research 998-999 (julho de 2014): 312–15. http://dx.doi.org/10.4028/www.scientific.net/amr.998-999.312.
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Purpose: The adult corneal epithelium is maintained by a population of limbal stem cells (LSCs), transmembrane protein prominin, regarded as stem cell marker was investigated on mouse corneal tissue, to study weather contains CD133-expressing cells and their distribution. Methods: Enucleated mouse eyes were embedded in OCT and cryosections were performed for mmunohistochemical studies using the avidin-biotin-peroxidase complex (ABC) procedure. Meanwhile, dissected mouse corneas were analyzed by westernblot. Results: In the adult mouse, 13A4 immunoreactivity was detected at the apical side of superficial corneal epithelium, including the limbus region, but not by stroma and endothelium. 115 KDa protein was approved in corneal tissue by Westernblot. Conclusions: The stem cell activity does not occur along the limbus but presumably presented by small portion of corneal epithelial cells which may hold a similar properties of stem cells.
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Liu, Cong, Xin Yu e Yun Kou. "Effect of diagnostic ultrasound on corneal apoptosis in rats". Tropical Journal of Pharmaceutical Research 19, n.º 9 (24 de novembro de 2020): 1947–51. http://dx.doi.org/10.4314/tjpr.v19i9.21.
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Purpose: To investigate the effect of diagnostic ultrasound on corneal apoptosis in rats.Methods: 24 male rats were randomly divided into 4 groups: control group, 10, 20 and 30 min group. The eyeballs of rats were irradiated continuously for different time lengths by Siemens ACUSON S2000 color Doppler ultrasound diagnostic instrument. 24 hours later, the animals were killed and the corneas were taken for Tunel apoptosis detection. The apoptosis rates of corneal epithelial cells, stromal cells and endothelial cells were calculated.Results: Apoptotic cells were detected in corneal epithelial cells, stromal cells and endothelial cells of normal rats. There was no significant difference between the 10 min group and the control group (P>0.05). The apoptosis rate of 20 min and 30 min groups was significantly higher than that of the control group. With the extension of irradiation time, the apoptosis rate of corneal epithelial cells, stromal cells and endothelial cells increased.Conclusion: 20 min of rat eyeball irradiated by diagnostic ultrasound can increase the apoptosis of corneal cells, and the apoptosis is aggravated with the prolongation of ultrasound irradiation time.
Keywords: Cornea; Ultrasonography; Apoptosis; Epithelial cells
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Notara, M., A. Lentzsch, M. Coroneo e C. Cursiefen. "The Role of Limbal Epithelial Stem Cells in Regulating Corneal (Lymph)angiogenic Privilege and the Micromilieu of the Limbal Niche following UV Exposure". Stem Cells International 2018 (2018): 1–15. http://dx.doi.org/10.1155/2018/8620172.
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The cornea is a clear structure, void of blood, and lymphatic vessels, functioning as our window to the world. Limbal epithelial stem cells, occupying the area between avascular cornea and vascularized conjunctiva, have been implicated in tissue border maintenance, preventing conjunctivalisation and propagation of blood and lymphatic vessels into the cornea. Defects in limbal epithelial stem cells are linked to corneal neovascularisation, including lymphangiogenesis, chronic inflammation, conjunctivalisation, epithelial abnormalities including the presence of goblet cells, breaks in Bowman’s membrane, persistent epithelial defects and ulceration, ocular surface squamous neoplasia, lipid keratopathy, pain, discomfort, and compromised vision. It has been postulated that pterygium is an example of focal limbal deficiency. Previous reports showing changes occurring in limbal epithelium during pterygium pathogenesis suggest that there is a link to stem cell damage. In this light, pterygium can serve as a model disease of UV-induced stem cell damage also characterised by corneal blood and lymphangiogenesis. This review focuses on the role of corneal and limbal epithelial cells and the stem cell niche in maintaining corneal avascularity and corneal immune privilege and how this may be deregulated following UV exposure. We present an overview of the PUBMED literature in the field as well as recent work from our laboratories.
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Krasnov, M. S., e V. P. Yamskova. "The Effect of Bioregulators Isolated from Blood Serum and Cornea of the Bovin’s Eye on the Condition of Tissue and Cells of the Rabbit Corneas during Cultivation and Storage". Ophthalmology in Russia 18, n.º 3 (1 de outubro de 2021): 488–94. http://dx.doi.org/10.18008/1816-5095-2021-3-488-494.
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Objective: to study the condition of the cornea, as well as its epithelial and endothelial cells, while maintaining in vitro at various temperature conditions, under the influence of a number of factors, including bioregulators isolated from blood serum and cornea of the bovine, and epidermal growth factor.Methods. The study was carried out on rabbit corneas stored at temperatures of +4, –86 °C, as well as the cultivation of endothelial and epithelial cells isolated from the cornea after storage at these temperatures, followed by histological and immunohistochemical studies.Results. Storage of the cornea at +4 °C for 10 days leads to corneal edema and significantly reduces their transparency, both bioregulators partially prevent a decrease in the transparency of the cornea, while the endothelial layer lyses in groups with the addition of epidermal growth factor and corneal bioregulator; but remains in the cornea with the addition of a serum bioregulator. All three factors contribute to the preservation of the Bowman membrane. In the corneas stored at –86 °C on the 30th day, a preserved endothelial layer was observed, and the epithelium retained its multilayering in all groups with the addition of factors other than the control group. In the control samples, the epithelial layer partially exfoliated, the endothelial layer was almost completely lysed. Both bioregulators stimulated the proliferation of cells isolated from the native cornea and enhanced the action of the epidermal growth factor. Similar results were obtained on cells isolated from stored corneas for 2 weeks at –86 °C. In the case of combined use of the epidermal growth factor and bioregulators on the 30th day, the endothelial layer was mainly preserved, the Descemet’s membrane was not broken. In the control samples, the epithelium was mainly single-layered, partially exfoliated, and the endothelial layer was completely lysed.Conclusion. Storage of cornea during hypothermia (+4 °С) does not provide corneal viability for longer than 10 days. Storage under conditions of cryopreservation (–86 °C) ensures the viability of the cornea for 60 days. Adding bioregulators and an epidermal growth factor to the basic preservation medium allows one to obtain a structurally safe and viable cornea, while all cellular layers of the cornea, including the endothelial layer, are preserved and viable.
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Morii, Tomoya, Takayoshi Sumioka, Ai Izutani-Kitano, Yukihisa Takada, Yuka Okada, Winston W. Y. Kao e Shizuya Saika. "A Case of Solitary Nonvascularized Corneal Epithelial Dysplasia". Case Reports in Ophthalmological Medicine 2016 (2016): 1–4. http://dx.doi.org/10.1155/2016/5687285.
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Background. Epithelial dysplasia is categorized as conjunctival/corneal intraepithelial neoplasia which is a precancerous lesion. The lesion is usually developed at the limbal region and grows towards central cornea in association with neovascularization into the lesion. Here, we report a case of isolated nonvascularized corneal epithelial dysplasia surrounded by normal corneal epithelium with immune histochemical finding of ocular surface tissues cytokeratins, for example, keratin 13 and keratin 12.Case Presentation. A 76-year-old man consulted us for visual disturbance with localized opacification of the corneal epithelium in his left eye. His visual acuity was 20/20 and 20/200 in his right and left eye, respectively. Slit lamp examination showed a whitish plaque-like lesion at the center of his left corneal epithelium. No vascular invasion to the lesion was found. The lesion was surgically removed and subjected to histopathological examination and diagnosed as epithelial dysplasia. Amyloidosis was excluded by direct fast scarlet 4BS (DFS) staining. Immunohistochemistry showed that the dysplastic epithelial cells express keratin 13 and vimentin, but not keratin 12, indicating that the neoplastic epithelial cells lacked corneal-type epithelium differentiation.Conclusions. The lesion was diagnosed as nonvascularized epithelial dysplasia of ocular surface. Etiology of the lesion is not known.
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Cui, Hao, Ying Liu e Yifei Huang. "Roles of TRIM32 in Corneal Epithelial Cells After Infection with Herpes Simplex Virus". Cellular Physiology and Biochemistry 43, n.º 2 (2017): 801–11. http://dx.doi.org/10.1159/000481563.
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Background: Epithelial cells play important roles as a critical barrier in protecting the cornea from microbial pathogens infection. Methods: In this study, we were aiming to investigate the role of E3 ubiquitin ligase tripartite motif protein 32 (TRIM32) in corneal epithelial cells in response to Herpes Simplex Virus type 1 (HSV-1) infection and to elucidate the underlying mechanisms. Results: We found the expression of TRIM32 was increased after infected with HSV-1 both in murine corneas and cultured human epithelial (HCE) cells. Furthermore, knockdown of the expression of TRIM32 significantly aggravated HSV-1 induced herpetic stromal keratitis (HSK) in mice and promoted the replication of HSV-1 in cultured HCE cells. We also observed that silencing of TRIM32 resulted in the decreased expression of IFN-β and suppressed activation of interferon regulatory factor 3 (IRF3) both in vivo and in vitro. Finally, we found TRIM32 positively regulate IFN-β production in corneal epithelial cells through promoting K63-linked polyubiquitination of stimulator of interferon genes (STING). Conclusion: In conclusion, our data suggested that TRIM32 as a crucial positive regulator of HSV-1 induced IFN-β production in corneal epithelial cells, and it played a predominant role in clearing HSV-1 from the cornea.
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Pedrotti, Emilio, Chiara Chierego, Tiziano Cozzini, Tommaso Merz, Neil Lagali, Alessandra De Gregorio, Adriano Fasolo, Erika Bonacci, Jacopo Bonetto e Giorgio Marchini. "In Vivo Confocal Microscopy of the Corneal-Conjunctival Transition in the Evaluation of Epithelial Renewal after SLET". Journal of Clinical Medicine 9, n.º 11 (6 de novembro de 2020): 3574. http://dx.doi.org/10.3390/jcm9113574.
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Examination of the corneal surface by in vivo confocal microscopy (IVCM) allows for objective identification of corneal and conjunctival cell phenotypes to evaluate different epithelialization patterns. Detection of a corneal-conjunctival epithelial transition could be considered as a sign of restored epithelial function following simple limbal epithelial transplantation (SLET). This is a prospective, interventional case series. We assessed patients with limbal stem cell deficiency (LSCD) by IVCM, preoperatively and at monthly intervals following SLET. Sectors in the central and peripheral cornea were scanned. Immediately upon detection of multi-layered cells with the epithelial phenotype in the central cornea and confirmation of epithelial transition in all corneal sectors, the decision for keratoplasty was taken. Ten patients were enrolled. After SLET, epithelial phenotype in the central cornea and an epithelial transition were identified within six and nine months in seven and one patients, respectively. One patient was a partial success and one failed. Five patients underwent keratoplasty, with stable results up to 12 months. Identification of the epithelial transition zone by IVCM permits assessment of the efficacy of SLET, enabling subsequent planning of keratoplasty for visual rehabilitation. The stability of the corneal surface following keratoplasty confirms that the renewal of the corneal epithelium was effectively retained.
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Sangwan, Virender S. "Limbal Stem Cells in Health and Disease". Bioscience Reports 21, n.º 4 (1 de agosto de 2001): 385–405. http://dx.doi.org/10.1023/a:1017935624867.
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Stem cells are present in all self-reviewing tissues and have unique properties. The ocular surface is made up of two distinct types of epithelial cells, constituting the conjunctival and the corneal epithelia. These epithelia are stratified, squamous and non-keratinized. Although anatomically continuous with each other at the corneoscleral limbus, the two cell phenotypes represent quite distinct subpopulations. The stem cells for the cornea are located at the limbus. The microenvironment of the limbus is considered to be important in maintaining stemness of the stem cells. They also act as a “barrier” to conjunctival epithelial cells and prevent them from migrating on to the corneal surface. In certain pathologic conditions, however, the limbal stem cells may be destroyed partially or completely resulting in varying degrees of stem cell deficiency with its characteristic clinical features. These include “conjunctivalization” of the cornea with vascularization, appearance of goblet cells, and an irregular and unstable epithelium. The stem cell deficiency can be managed with auto or allotransplantation of these cells. With the latter option, systemic immunosuppression is required. The stem cells can be expanded ex vivo on a processed human amniotic membrane and transplanted back to ocular surface with stem cell deficiency without the need of immunosuppression.
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Nguyen, Hong Thi, Kasem Theerakittayakorn, Sirilak Somredngan, Apichart Ngernsoungnern, Piyada Ngernsoungnern, Pishyaporn Sritangos, Mariena Ketudat-Cairns et al. "Signaling Pathways Impact on Induction of Corneal Epithelial-like Cells Derived from Human Wharton’s Jelly Mesenchymal Stem Cells". International Journal of Molecular Sciences 23, n.º 6 (12 de março de 2022): 3078. http://dx.doi.org/10.3390/ijms23063078.
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Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of β-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-β signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.
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Xie, Ruo Zhong, Serina Stretton e Deborah F. Sweeney. "Artificial Cornea: Towards a Synthetic Onlay for Correction of Refractive Error". Bioscience Reports 21, n.º 4 (1 de agosto de 2001): 513–36. http://dx.doi.org/10.1023/a:1017900111663.
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Synthetic onlays that are implanted onto the surface of the cornea have the potential to become an alternative to spectacles and contact lenses for the correction of refractive error. A successful corneal onlay is dependent on development of a biocompatible polymer material that will maintain a healthy cornea after implantation and that will promote growth of corneal epithelial cells over the onlay, and development of a method for attachment of the onlay with minimal surgical invasiveness. The ideal onlay should be made of a material that is highly permeable yet has sufficient surface characteristics to stimulate stable and firm attachment of the corneal epithelium over the onlay. Recent research indicates that collagen I coated polymer materials that mimic the basement membrane of the corneal epithelium promote the most favorable growth of epithelial cells in vivo in comparison to wholly biological or synthetic materials.
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Lee, Hyo Kyung, Jin Suk Ryu, Hyun Jeong Jeong, Mee Kum Kim e Joo Youn Oh. "Protection of Corneal Limbus from Riboflavin Prevents Epithelial Stem Cell Loss after Collagen Cross-Linking". Journal of Ophthalmology 2018 (3 de junho de 2018): 1–7. http://dx.doi.org/10.1155/2018/6854298.
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Purpose. To investigate whether the protection of corneal limbus from riboflavin exposure during collagen cross-linking (CXL) prevents limbal epithelial stem cell (LESC) loss. Methods. Ten New Zealand white rabbits received an epithelium-off CXL using an accelerated protocol. Seven days before procedure, 5-bromo-2-deoxyuridine (BrdU) was intraperitoneally injected. During procedure, riboflavin was applied to the corneal surface within a 9 mm diameter retention ring in 5 rabbits, thereby preventing the limbus from riboflavin exposure. In other 5 rabbits, riboflavin was instilled every 2 min, allowing the spillover to the limbus. One day after UVA irradiation, corneas were subjected to histological and molecular assays. Results. There were no differences in corneal thickness and epithelial healing between the groups. The numbers of BrdU-labelled and p63+ limbal epithelial cells were markedly reduced in the group without a ring, but significantly increased when a ring was used. Robust expression of CK3/12 was observed in the limbal epithelium in the group with a ring. The mRNA levels of ABCG2, FGF2, IL-1β, and IL-6 were significantly increased in the corneas with a ring. Conclusions. Protection of limbus from riboflavin during CXL was effective in preserving LESCs. However, inflammation was increased in the cornea treated with riboflavin using a ring.
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Leher, Henry, Robert Silvany, Hassan Alizadeh, Jing Huang e Jerry Y. Niederkorn. "Mannose Induces the Release of Cytopathic Factors from Acanthamoeba castellanii". Infection and Immunity 66, n.º 1 (1 de janeiro de 1998): 5–10. http://dx.doi.org/10.1128/iai.66.1.5-10.1998.
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ABSTRACT Acanthamoeba keratitis is a chronic inflammatory disease of the cornea which is highly resistant to many antimicrobial agents. The pathogenic mechanisms of this disease are poorly understood. However, it is believed that the initial phases in the pathogenesis of Acanthamoeba keratitis involve parasite binding and lysis of the corneal epithelium. These processes were examined in vitro, usingAcanthamoeba castellanii trophozoites. Parasites readily adhered to Chinese hamster corneal epithelial cells in vitro; however, parasite binding was strongly inhibited by mannose but not by lactose. Although mannose prevented trophozoite binding, it did not affect cytolysis of corneal epithelial cells. Moreover, mannose treatment induced trophozoites to release cytolytic factors that lysed corneal epithelial cells in vitro. These factors were uniquely induced by mannose because supernatants collected from either untreated trophozoites or trophozoites treated with other sugars failed to lyse corneal cells. The soluble factors were size fractionated in centrifugal concentrators and found to be ≥100 kDa. Treatment of the supernatants with the serine protease inhibitor phenylmethylsulfonyl fluoride inhibited most, but not all, of the cytopathic activity. These data suggest that the binding ofAcanthamoeba to mannosylated proteins on the corneal epithelium may exacerbate the pathogenic cascade by initiating the release of cytolytic factors.
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Saban, Daniel, Taakaki Hattori, Sunil Chauhan e Reza Dana. "Identification and characterization of langerin positive dendritic cells in the normal cornea and in transplantation. (145.27)". Journal of Immunology 184, n.º 1_Supplement (1 de abril de 2010): 145.27. http://dx.doi.org/10.4049/jimmunol.184.supp.145.27.
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Abstract A novel population of migratory dendritic cells (DCs), distinct from Langerhans cells (LC), was recently found to which also express langerin (CD207+) and reside throughout the body (skin, lung, gut, and liver). Moreover, such CD207+ DCs were shown to be important in generating immunity. Thus, we investigated whether CD207+ DCs exist in the cornea and their possible migration to lymph nodes (LN) in corneal alloimmunity. Methods: C57BL/6 corneas (n=30) were collected and epithelium separated from stroma. Tissue was pooled separately and digested enzymatically for FACS analysis of CD45, CD11c, CD207, CD205, and CD103. Immunohistology was also examined. Furthermore, BALB/c mice were allografted with C57BL/6 corneas and LNs evaluated for corneal CD207+ DCs vs. LCs. Results: Most epithelial and stromal DCs were CD207+ and co-expressed CD205+, but only the stromal subset expressed CD103+. Interestingly, following allotransplantation, the frequency of the stromal DCs (CD11c+ CD8a- CD207+ CD103+) decreased dramatically (by 4-fold) in ipsilateral LNs relative to naïve mice; whereas, the epithelial DCs (CD103-) markedly increased. A similar, but much less dramatic, trend was seen in the contralateral LN. Conclusions: This indicates the presence of CD207+ DCs in the cornea, distinct from LCs of the epithelium. Interestingly, a dramatic and diametrically opposed flux of such stromal DCs vs. LCs to LNs following transplantation suggests potentially differing functions in alloimmunity.
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Kacham, Santhosh, Tejal Sunil Bhure, Sindhuja D. Eswaramoorthy, Gaurav Naik, Subha Narayan Rath, Sreenivasa Rao Parcha, Sayan Basu, Virender Singh Sangwan e Sachin Shukla. "Human Umbilical Cord-Derived Mesenchymal Stem Cells Promote Corneal Epithelial Repair In Vitro". Cells 10, n.º 5 (19 de maio de 2021): 1254. http://dx.doi.org/10.3390/cells10051254.
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Corneal injuries are among the leading causes of blindness and vision impairment. Trauma, infectious keratitis, thermal and chemical (acids and alkali burn) injuries may lead to irreversible corneal scarring, neovascularization, conjunctivalization, and limbal stem cell deficiency. Bilateral blindness constitutes 12% of total global blindness and corneal transplantation remains a stand-alone treatment modality for the majority of end-stage corneal diseases. However, global shortage of donor corneas, the potential risk of graft rejection, and severe side effects arising from long-term use of immunosuppressive medications, demands alternative therapeutic approaches. Umbilical cord-derived mesenchymal stem cells can be isolated in large numbers using a relatively less invasive procedure. However, their role in injury induced corneal repair is largely unexplored. Here, we isolated, cultured and characterized mesenchymal stem cells from human umbilical cord, and studied the expression of mesenchymal (CD73, CD90, CD105, and CD34), ocular surface and epithelial (PAX6, WNT7A, and CK-8/18) lineage markers through immunofluorescence. The cultured human limbal and corneal epithelial cells were used as controls. Scratch assay was used to study the corneal epithelial repair potential of umbilical cord-derived mesenchymal stem cells, in vitro. The in vitro cultured umbilical cord-derived mesenchymal stem cells were plastic adherent, showed trilineage differentiation and expressed: mesenchymal markers CD90, CD105, CD73; epithelial marker CK-8/18, and ocular lineage developmental markers PAX6 and WNT-7A. Our findings suggest that umbilical cord-derived mesenchymal stem cells promote repair of the injured corneal epithelium by stimulating the proliferation of corneal epithelial cells, in vitro. They may serve as a potential non-ocular source of stem cells for treating injury induced bilateral corneal diseases.
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Ramos, Tiago, Deborah Scott e Sajjad Ahmad. "An Update on Ocular Surface Epithelial Stem Cells: Cornea and Conjunctiva". Stem Cells International 2015 (2015): 1–7. http://dx.doi.org/10.1155/2015/601731.
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The human ocular surface (front surface of the eye) is formed by two different types of epithelia: the corneal epithelium centrally and the conjunctival epithelium that surrounds this. These two epithelia are maintained by different stem cell populations (limbal stem cells for the corneal epithelium and the conjunctival epithelial stem cells). In this review, we provide an update on our understanding of these epithelia and their stem cells systems, including embryology, new markers, and controversy around the location of these stem cells. We also provide an update on the translation of this understanding into clinical applications for the treatment of debilitating ocular surface diseases.
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Omaña-Molina, Maritza, Fernando Navarro-García, Arturo González-Robles, José de Jesús Serrano-Luna, Rafael Campos-Rodríguez, Adolfo Martínez-Palomo, Víctor Tsutsumi e Mineko Shibayama. "Induction of Morphological and Electrophysiological Changes in Hamster Cornea after In Vitro Interaction with Trophozoites of Acanthamoeba spp." Infection and Immunity 72, n.º 6 (junho de 2004): 3245–51. http://dx.doi.org/10.1128/iai.72.6.3245-3251.2004.
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ABSTRACT Acanthamoeba castellani and Acanthamoeba polyphaga are free-living amebae that cause keratitis and granulomatous encephalitis in humans. We have analyzed the early morphological and electrophysiological changes occurring during the in vitro interaction of cultured amebae with intact or physically damaged corneas obtained from hamsters. Both species of Acanthamoeba produced similar cytopathic changes, as seen by light microscopy and scanning electron microscopy. After adhesion to the epithelial surface, trophozoites formed clumps and migrated toward the cell borders, causing the separation of adjacent cells at 1 h of coculture. At later stages (2 to 4 h), some amebae were found under desquamating epithelial cells whereas others were seen associated with damaged cells or forming amebostome-like structures to ingest detached epithelial cells. Control corneas incubated in culture medium conditioned with amebae showed a cytoplasmic vacuolization and blurring of the epithelial-stromal junction. The early stages of corneal epithelial damage caused by amebae were also analyzed by measuring the transepithelial resistance changes in corneas mounted in Ussing chambers. Both species of Acanthamoeba caused a rapid decrease in electrical resistance. The present observations demonstrate that under in vitro conditions, Acanthamoeba trophozoites rapidly cause significant damage to the corneal epithelium. Furthermore, in our experimental model, previous physical damage to the corneas was not a prerequisite for the development of amebic corneal ulcerations.
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Fu, Meng-Jun, Shan Duan, Hao-Run Zhang, Jing-Jing Zhao, Li Zeng, Rui Wang e Xing-Tao Zhou. "Corneal histomorphology and electron microscopic observation of R124L mutated corneal dystrophy in a relapsed pedigree". International Journal of Ophthalmology 15, n.º 9 (18 de setembro de 2022): 1416–22. http://dx.doi.org/10.18240/ijo.2022.09.02.
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AIM: To investigate the histological characteristics and ultrastructure of recurrent Chinese R124L mutated corneal dystrophy after keratoplasty. METHODS: The subjects were enrolled from a Chinese family of corneal dystrophy with R124L heterozygous gene mutation and with a history of consanguineous marriage. Normal corneal samples were used as controls. RESULTS: In this family, 2 patients (3 eyes) underwent penetrating keratoplasty (PKP) and 2 patients (4 eyes) underwent lamellar keratoplasty (LKP). They had recurrence at 33.5±3.0 (range 30-36)mo after keratoplasty. Among them, 1 patient (1 eye) underwent PKP again and 1 patient (2 eyes) underwent LKP again. In the R124L mutated recurrent corneal dystrophy, the corneal turbidity was mainly distributed from the upper corneal cortex to the anterior stroma; the corneal epithelium surface was rougher and more uneven; and, the corneal erosions were larger. Hematoxylin-eosin staining showed that the thickness of the corneal epithelium was uneven; the arrangement of the epithelial cells was disordered; and, some corneal epithelial cells were swollen. The results of Congo red staining, Masson’s trichrome staining and Periodic acid-Schiff staining were positive, while that of Alcian blue staining was negative. Under a transmission electron microscope, deposition of high electron density substances between epithelial and basal cells, and, apoptosis of basal cells were observed. Many high electron density depositions were observed in the sub-epithelial and anterior corneal matrix. CONCLUSION: In the Chinese family of recurrent corneal dystrophy with R124L gene mutation, the corneal epithelia of the recurrent cases are rougher, and the corneal depositions are extracellular amyloid fibrin.
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Fukuoka, Hideki, Satoshi Kawasaki, Norihiko Yokoi, Kenta Yamasaki e Shigeru Kinoshita. "Cytopathological Features of a Severe Type of Corneal Intraepithelial Neoplasia". Case Reports in Ophthalmology 7, n.º 1 (11 de maio de 2016): 253–61. http://dx.doi.org/10.1159/000445937.
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Purpose: To report the cytopathological features of corneal intraepithelial neoplasia (CIN) through the investigation of cytokeratin expression pattern, keratinization, cell proliferation, apoptosis, and epithelial mesenchymal transition. Patient and Methods: Corneal tissue excised from a CIN patient was examined in this study. Cryosections of the excised CIN epithelial tissue were examined by immunostaining analysis using antibodies against cytokeratins, keratinization-related proteins, Ki-67, human telomerase reverse transcriptase (hTERT), and epithelial mesenchymal transition (EMT)-related proteins. Subcellular localization of F-actin was also analyzed using phalloidin. For the detection of apoptotic cells, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed. Real-time polymerase chain reaction was performed to quantify the expression level of hTERT in the CIN epithelium. Results: The CIN epithelium exhibited a significantly altered cytokeratin expression pattern compared to normal corneas with an upregulated expression of keratinization-related proteins. The CIN epithelium also demonstrated an increased number of Ki-67-positive cells with an upregulated expression of hTERT, while exhibiting an increased number of apoptotic cells. EMT did not occur in the CIN epithelium. Conclusion: CIN epithelium seems to be slightly dedifferentiated from the corneal epithelial lineage. The status of cell proliferation and apoptosis in the CIN epithelium was significantly altered from that of normal corneal epithelium, but its malignancy level does not appear to be as high as that of metastasis-competent malignant cancers.
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Huhtala, Anne, Sami K. Nurmi, Hanna Tähti, Lotta Salminen, Päivi Alajuuma, Immo Rantala, Heikki Helin e Hannu Uusitalo. "The Immunohistochemical Characterisation of an SV40-immortalised Human Corneal Epithelial Cell Line". Alternatives to Laboratory Animals 31, n.º 4 (julho de 2003): 409–17. http://dx.doi.org/10.1177/026119290303100407.
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Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.
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McKay, Tina B., Audrey E. K. Hutcheon, James D. Zieske e Joseph B. Ciolino. "Extracellular Vesicles Secreted by Corneal Epithelial Cells Promote Myofibroblast Differentiation". Cells 9, n.º 5 (26 de abril de 2020): 1080. http://dx.doi.org/10.3390/cells9051080.
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The corneal epithelium mediates the initial response to injury of the ocular surface and secretes a number of profibrotic factors that promote corneal scar development within the stroma. Previous studies have shown that corneal epithelial cells also secrete small extracellular vesicles (EVs) in response to corneal wounding. In this paper, we hypothesized that EVs released from corneal epithelial cells in vitro contain protein cargo that promotes myofibroblast differentiation, the key cell responsible for scar development. We focused on the interplay between corneal epithelial-derived EVs and the stroma to determine if the corneal fibroblast phenotype, contraction, proliferation, or migration were promoted following vesicle uptake by corneal fibroblasts. Our results showed an increase in myofibroblast differentiation based on α-smooth muscle actin expression and elevated contractility following EV treatment compared to controls. Furthermore, we characterized the contents of epithelial cell-derived EVs using proteomic analysis and identified the presence of provisional matrix proteins, fibronectin and thrombospondin-1, as the dominant encapsulated protein cargo secreted by corneal epithelial cells in vitro. Proteins associated with the regulation of protein translation were also abundant in EVs. This paper reveals a novel role and function of EVs secreted by the corneal epithelium that may contribute to corneal scarring.
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Theerakittayakorn, Kasem, Hong Thi Nguyen, Jidapa Musika, Hataiwan Kunkanjanawan, Sumeth Imsoonthornruksa, Sirilak Somredngan, Mariena Ketudat-Cairns e Rangsun Parnpai. "Differentiation Induction of Human Stem Cells for Corneal Epithelial Regeneration". International Journal of Molecular Sciences 21, n.º 21 (22 de outubro de 2020): 7834. http://dx.doi.org/10.3390/ijms21217834.
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Deficiency of corneal epithelium causes vision impairment or blindness in severe cases. Transplantation of corneal epithelial cells is an effective treatment but the availability of the tissue source for those cells is inadequate. Stem cells can be induced to differentiate to corneal epithelial cells and used in the treatment. Multipotent stem cells (mesenchymal stem cells) and pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are promising cells to address the problem. Various protocols have been developed to induce differentiation of the stem cells into corneal epithelial cells. The feasibility and efficacy of both human stem cells and animal stem cells have been investigated for corneal epithelium regeneration. However, some physiological aspects of animal stem cells are different from those of human stem cells, the protocols suited for animal stem cells might not be suitable for human stem cells. Therefore, in this review, only the investigations of corneal epithelial differentiation of human stem cells are taken into account. The available protocols for inducing the differentiation of human stem cells into corneal epithelial cells are gathered and compared. Also, the pathways involving in the differentiation are provided to elucidate the relevant mechanisms.
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Sun, Tung-Tien. "Altered phenotype of cultured urothelial and other stratified epithelial cells: implications for wound healing". American Journal of Physiology-Renal Physiology 291, n.º 1 (julho de 2006): F9—F21. http://dx.doi.org/10.1152/ajprenal.00035.2006.
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The differentiation of cultured stratified epithelial cells can deviate significantly from that of normal epithelium, leading to suggestions that cultured cells undergo abnormal differentiation, or a truncated differentiation. Thus cultured epidermal and corneal epithelial cells stop synthesizing their tissue-specific keratin pair K1/K10 and K3/K12, respectively. The replacement of these keratins in the suprabasal compartment by K6/K16 keratins that are made by all stratified squamous epithelia during hyperplasia rules out a truncated differentiation. Importantly, the keratin pattern of in vivo corneal epithelium undergoing wound repair mimics that of cultured rabbit corneal epithelial cells. Although cultured urothelial cells continue to synthesize uroplakins, which normally form two-dimensional crystalline urothelial plaques covering almost the entire apical urothelial surface, these proteins do not assemble into crystals in cultured cells. Cultured epithelial cells can, however, rapidly regain normal differentiation on the removal of mitogenic stimuli, the use of a suitable extracellular matrix, or the transplantation of the cells to an in vivo, nonmitogenic environment. These data suggest that cultured epithelial cells adopt altered differentiation patterns mimicking in vivo regenerating or hyperplastic epithelia. Blocking the synthesis of tissue-specific differentiation products, such as the K1 and K10 keratins designed to form extensive disulfide cross-links in cornified cells, or the assembly of uroplakin plaques allows epithelial cells to better migrate and proliferate, activities that are of overriding importance during wound repair. Cultured urothelial and other stratified epithelial cells provide excellent models for studying the regulation of the synthesis and assembly of differentiation products, a key cellular process during epithelial wound repair.
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Milyudin, E. S., e D. A. Drozdetskaya. "MEOHANisMs oF HEALiNG oF OoRNEAL DEFEOTs wiTH THE USE OF SiLiOA DESiCOATiON AMNiOTiO MEMBRANE". Aspirantskiy Vestnik Povolzhiya 19, n.º 1-2 (15 de março de 2019): 80–84. http://dx.doi.org/10.17816/2072-2354.2019.19.1.80-84.
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The purpose of this work is to study the mechanism of healing of corneal epithelium defect by using silica desiccation amniotic membrane at the cytomorphological level. 7 patients of Samara Regional Clinical Ophtalmologic Hospital named after T.I. Eroshevsky formed the main group of the perforemd study. They were diagnosed with recurrent trophic ulcers of the epithelium and corneal stroma. Their corneal defects were covered with the silica desiccation amniotic membrane. A control group included 5 patients with the same diagnosis. They were observed and treated without amniotic membrane application. Control over the regeneration of corneal defects was carried out by biomicroscopy with fluorescein staining, as well as by cytological examination of impression smears from the surface of the cornea on the 1st, 7th and 15th day after the treatment onset. Patient were being followed up for 6 months. Results. Cytomorphological examination on the 1st day after covering the cornea with the amniotic membrane showed the presence of separate long cells of irregular shape. Both layers of smaller epithelial cells and multiple neutrophils were revealed in the control group. By the 7th day cytograms of the patients in the main group had shown mature cells, impression smears in patients of the control group demonstrated epithelial cells of round shape with darkened cytoplasm and a large number of inclusions. On the 15th day cytograms of patients of both groups almost did not differ. During the following 6 months’ observation recurrent corneal epithelial defects with the inflammatory process were noted in patients of the control group. The main group didn’t show such conditions. Conclusions. Silica desiccation amniotic membrane contributes to the increase in the number of epithelial cells, movement and their firm fixation on the corneal surface as well as reduction of the inflammatory process.
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Oliva, Joan, Fawzia Bardag-Gorce e Yutaka Niihara. "Clinical Trials of Limbal Stem Cell Deficiency Treated with Oral Mucosal Epithelial Cells". International Journal of Molecular Sciences 21, n.º 2 (9 de janeiro de 2020): 411. http://dx.doi.org/10.3390/ijms21020411.
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The corneal surface is an essential organ necessary for vision, and its clarity must be maintained. The corneal epithelium is renewed by limbal stem cells, located in the limbus and in palisades of Vogt. Palisades of Vogt maintain the clearness of the corneal epithelium by blocking the growth of conjunctival epithelium and the invasion of blood vessels over the cornea. The limbal region can be damaged by chemical burns, physical damage (e.g., by contact lenses), congenital disease, chronic inflammation, or limbal surgeries. The degree of limbus damage is associated with the degree of limbal stem cells deficiency (partial or total). For a long time, the only treatment to restore vision was grafting part of the healthy cornea from the other eye of the patient or by transplanting a cornea from cadavers. The regenerative medicine and stem cell therapies have been applied to restore normal vision using different methodologies. The source of stem cells varies from embryonic stem cells, mesenchymal stem cells, to induced pluripotent stem cells. This review focuses on the use of oral mucosa epithelial stem cells and their use in engineering cell sheets to treat limbal stem cell deficient patients.
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Cai, Cindy X., David E. Birk e Thomas F. Linsenmayer. "Nuclear Ferritin Protects DNA From UV Damage in Corneal Epithelial Cells". Molecular Biology of the Cell 9, n.º 5 (maio de 1998): 1037–51. http://dx.doi.org/10.1091/mbc.9.5.1037.
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Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3′-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.
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Shen, Yun-Zhi, Mi Xu e Song Sun. "In Vivo Confocal Microscopy Observation of Cell and Nerve Density in Different Corneal Regions with Monocular Pterygium". Journal of Ophthalmology 2020 (23 de março de 2020): 1–7. http://dx.doi.org/10.1155/2020/6506134.
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Purpose. To investigate the effects of pterygium on corneal cell and nerve density in patients with unilateral pterygium using in vivo laser scanning confocal microscopy (LSCM). Methods. In this cross-sectional study, 24 patients with unilateral pterygium who were treated in the Department of Ophthalmology of the Second People’s Hospital of Wuxi City from April 2018 to July 2018 were analyzed. Each eye with pterygium and its fellow eye were imaged by LSCM. The density of basal corneal epithelial cells, anterior stromal cells, posterior stromal cells, dendritic cells, and endothelial cells in pterygium and adjacent clear cornea was measured. In the fellow eyes, the central cornea, nasal cornea, nasal mid-peripheral cornea, and temporal cornea were imaged. The difference in the density of cells and subepithelial nerve fibers in different corneal regions of eyes with pterygium was analyzed. The cell and nerve density of the fellow cornea were also measured to exclude the influencing factors. Results. The density of corneal basal epithelial cells in the central corneas of eyes with pterygium was 6497 ± 1776 cells/mm2, which was higher than that in the area near the head of pterygium (5580 ± 1294 cells/mm2, P<0.001), the region above pterygium (6097 ± 1281 cells/mm2, P=0.049), and the region below pterygium (5463 ± 1007 cells/mm2, P=0.001). The density of anterior stromal cells in the central cornea was 742 ± 243 cells/mm2, which was higher than that in the area near the head of pterygium (587 ± 189 cells/mm2, P=0.005), the region below pterygium (492 ± 159 cells/mm2, P=0.005), and the temporal cornea (574 ± 164 cells/mm2, P=0.003). The density of endothelial cells in the central cornea was 2398 ± 260 cells/mm2, which was higher than that in the area near the head of pterygium (2296 ± 231 cells/mm2, P=0.011) and the region below pterygium (2272 ± 400 cells/mm2, P=0.020). The density of dendritic cells in the central cornea was 53 ± 48 cells/mm2, which was lower than that in the area near the head of pterygium (250 ± 224 cells/mm2, P=0.001), the upper region (103 ± 47 cells/mm2, P=0.006), and the lower region (90 ± 48 cells/mm2, P=0.023). The corneal nerve fiber length (CNFL) in the center was higher than that in the area near the head of pterygium, the upper region, and the lower region. Compared with fellow eyes, eyes with pterygium had a significantly higher mean corneal power (KM) (P<0.001). There was a significant positive linear relationship between the corneal area invaded by pterygium of pterygia and KM (r = 0.609, P=0.009). Conclusion. Basal epithelial cells, stromal cells, endothelial cells, dendritic cells, and subepithelial nerve fibers in the central cornea of eyes with pterygium were different from those of pterygium and adjacent clear cornea. LSCM is effective for observing the morphology and quantity of corneal cells in pterygium.
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Cejka, Cestmir, Vladimir Holan, Peter Trosan, Alena Zajicova, Eliska Javorkova e Jitka Cejkova. "The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns". Oxidative Medicine and Cellular Longevity 2016 (2016): 1–12. http://dx.doi.org/10.1155/2016/5843809.
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The aim of this study was to examine whether mesenchymal stem cells (MSCs) and/or corneal limbal epithelial stem cells (LSCs) influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH) were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs), with adipose tissue MSCs (Ad-MSCs), or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS), α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and vascular endothelial factor (VEGF) were low. The central corneal thickness (taken as an index of corneal hydration) increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-β1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties.
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Borzenok, S. A., B. E. Malyugin, M. Yu Gerasimov e D. S. Ostrovsky. "Cultivated autologous oral mucosal epithelial transplantation". Russian Journal of Transplantology and Artificial Organs 23, n.º 1 (10 de abril de 2021): 171–77. http://dx.doi.org/10.15825/1995-1191-2021-1-171-177.
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According to the World Health Organization, corneal blindness is the fourth most common cause of blindness and visual impairment worldwide. In Russia, up to 18% of blindness is caused by corneal damage. Limbal stem cell deficiency (LSCD) is one of the causes of corneal blindness and visual impairment due to anterior epithelial replacement with fibrovascular pannus. Bilateral LSCD may develop in patients with aniridia, Steven-Jones syndrome, and severe corneal burns of both eyes, leading to severe decrease in visual acuity in both eyes and, as a consequence, physical disability associated with blindness. In such cases, cell therapy, based on autologous oral epithelial culture as an alternative to allogeneic limbus transplants, is proposed for reconstruction of the anterior corneal epithelium. This new treatment method promotes corneal reepithelization, better visual acuity, reduced nonspecific ocular complaints and improved quality of life of patients. The effectiveness and significant increase in the frequency of transparent engraftment of donor corneas after cell therapy drives huge interest in this topic all over the world. This review presents literature data on the features of histotopography and methods for obtaining a cultured autologous oral mucosal epithelium, on cell markers that are used to identify epithelial cells, and on methods for creating cell grafts for subsequent transplantation to the corneal surface in LSCD patients.
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Jester, J. V., T. Moller-Pedersen, J. Huang, C. M. Sax, W. T. Kays, H. D. Cavangh, W. M. Petroll e J. Piatigorsky. "The cellular basis of corneal transparency: evidence for ‘corneal crystallins’". Journal of Cell Science 112, n.º 5 (1 de março de 1999): 613–22. http://dx.doi.org/10.1242/jcs.112.5.613.
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In vivo corneal light scattering measurements using a novel confocal microscope demonstrated greatly increased backscatter from corneal stromal fibrocytes (keratocytes) in opaque compared to transparent corneal tissue in both humans and rabbits. Additionally, two water-soluble proteins, transketolase (TKT) and aldehyde dehydrogenase class 1 (ALDH1), isolated from rabbit keratocytes showed unexpectedly abundant expression (approximately 30% of the soluble protein) in transparent corneas and markedly reduced levels in opaque scleral fibroblasts or keratocytes from hazy, freeze injured regions of the cornea. Together these data suggest that the relatively high expressions of TKT and ALDH1 contribute to corneal transparency in the rabbit at the cellular level, reminiscent of enzyme-crystallins in the lens. We also note that ALDH1 accumulates in the rabbit corneal epithelial cells, rather than ALDH3 as seen in other mammals, consistent with the taxon-specificity observed among lens enzyme-crystallins. Our results suggest that corneal cells, like lens cells, may preferentially express water-soluble proteins, often enzymes, for controlling their optical properties.
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Soleimani, Mohammad, Kasra Cheraqpour, Raghuram Koganti, Seyed Mahbod Baharnoori e Ali R. Djalilian. "Concise Review: Bioengineering of Limbal Stem Cell Niche". Bioengineering 10, n.º 1 (12 de janeiro de 2023): 111. http://dx.doi.org/10.3390/bioengineering10010111.
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The corneal epithelium is composed of nonkeratinized stratified squamous cells and has a significant turnover rate. Limbal integrity is vital to maintain the clarity and avascularity of the cornea as well as regeneration of the corneal epithelium. Limbal epithelial stem cells (LESCs) are located in the basal epithelial layer of the limbus and preserve this homeostasis. Proper functioning of LESCs is dependent on a specific microenvironment, known as the limbal stem cell niche (LSCN). This structure is made up of various cells, an extracellular matrix (ECM), and signaling molecules. Different etiologies may damage the LSCN, leading to limbal stem cell deficiency (LSCD), which is characterized by conjunctivalization of the cornea. In this review, we first summarize the basics of the LSCN and then focus on current and emerging bioengineering strategies for LSCN restoration to combat LSCD.
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Sun, Yan, Amy G. Hise, Carolyn M. Kalsow e Eric Pearlman. "Staphylococcus aureus-Induced Corneal Inflammation Is Dependent on Toll-Like Receptor 2 and Myeloid Differentiation Factor 88". Infection and Immunity 74, n.º 9 (setembro de 2006): 5325–32. http://dx.doi.org/10.1128/iai.00645-06.
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ABSTRACT Toll-like receptors (TLRs) expressed by the corneal epithelium represent a first line of host defense to microbial keratitis. The current study examined the role of TLR2, TLR4, and TLR9 and the common adaptor molecule myeloid differentiation factor 88 (MyD88) in a Staphylococcus aureus model of corneal inflammation. The corneal epithelia of C57BL/6, TLR2−/−, TLR4−/−, TLR9−/−, and MyD88−/− mice were abraded using a trephine and epithelial brush and were exposed to heat- or UV-inactivated S. aureus clinical strain 8325-4 and other clinical isolates. Corneal thickness and haze were measured by in vivo confocal microscopy, neutrophil recruitment to the corneal stroma was quantified by immunohistochemistry, and cytokine production was measured by enzyme-linked immunosorbent assay. The exposure of corneal epithelium to S. aureus induced neutrophil recruitment to the corneal stroma and increased corneal thickness and haze in control C57BL/6 mice but not in TLR2−/− or MyD88−/− mice. The responses of TLR4−/− and TLR9−/− mice were similar to those of C57BL/6 mice. S. aureus-induced cytokine production by corneal epithelial cells and neutrophils was also significantly reduced in TLR2−/− mice compared with that in C57BL/6 mice. These findings indicate that S. aureus-induced corneal inflammation is mediated by TLR2 and MyD88 in resident epithelial cells and infiltrating neutrophils.
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Das, Prosun, Jacintha Archana Pereira, Malay Chaklader, Aditya Law, Ketaki Bagchi, Gautam Bhaduri, Samaresh Chaudhuri e Sujata Law. "Phenotypic alteration of limbal niche–associated limbal epithelial stem cell deficiency by ultraviolet-B exposure–induced phototoxicity in mice". Biochemistry and Cell Biology 91, n.º 3 (junho de 2013): 165–75. http://dx.doi.org/10.1139/bcb-2012-0082.
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Good vision requires a healthy cornea, and a healthy cornea needs healthy stem cells. Limbal epithelial stem cells (LESCs) are a traditional source of corneal epithelial cells and are recruited for the continuous production of epithelium without seizing throughout an animal's life, which maintains corneal transparency. Like the maintenance of other adult somatic stem cells, the maintenance of LESCs depends on the specific microenvironmental niche in which they reside. The purpose of this study was to determine the microenvironmental damage associated with LESCs fate due to ultraviolet (UV)-B exposure in a mouse model. Structural alteration and deregulation of the stem cell and its neighboring niche components were observed by using clinical, morphological, explant culture study, and flowcytometric analysis, which demonstrated that the limbal microenvironment plays an important role in cornea-related disease development. In UV-exposed mice, overexpression of vascular endothelial growth factor receptor 2 indicated neovascularization, decreased CD38 expression signified the alteration of limbal epithelial superficial cells, and the loss of limbal stem cell marker p63 indicated limbal stem cell deficiency in the limbal vicinity. We concluded that LESC deficiency diseases (LESCDDs) are associated with pathophysiological changes in the LESC niche, with some inhibitory interception such as UV-B irradiation, which results in corneal defects.
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Le-Bel, Gaëtan, Sergio Cortez Ghio, Louis-Philippe Guérin, Francis Bisson, Lucie Germain e Sylvain L. Guérin. "Irradiated Human Fibroblasts as a Substitute Feeder Layer to Irradiated Mouse 3T3 for the Culture of Human Corneal Epithelial Cells: Impact on the Stability of the Transcription Factors Sp1 and NFI". International Journal of Molecular Sciences 20, n.º 24 (13 de dezembro de 2019): 6296. http://dx.doi.org/10.3390/ijms20246296.
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Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. In this study, we investigated the impact of replacing irradiated 3T3 (i3T3) murine fibroblast feeder cells by irradiated human corneal fibroblasts (iHFL) on the expression of Sp1 and NFI and evaluated their contribution to the proliferative properties of human corneal epithelial cells (hCECs) in both monolayer cultures and human tissue engineered corneas (hTECs). hCECs co-cultured with iHFL could be maintained for up to two more passages than when they were grown with i3T3. Western Blot and electrophoretic mobility shift assays (EMSAs) revealed no significant difference in the feeder-layer dependent increase in Sp1 at both the protein and DNA binding level, respectively, between HCECs grown with either i3T3 or iHFL. On the other hand, a significant increase in the expression and DNA binding of NFI was observed at each subsequent passage when hCECs were co-cultured along with i3T3. These changes were found to result from an increased expression of the NFIA and NFIB isoforms in hCECs grown with i3T3. Exposure of hCECs to cycloheximide revealed an increased stability of NFIB that likely resulted from post-translational glycosylation of this protein when these cells were co-cultured with i3T3. In addition, iHFL were as efficient as i3T3 at preserving corneal, slow-cycling, epithelial stem cells in the basal epithelium of the reconstructed hTECs. Furthermore, we observed an increased expression of genes whose encoded products promote hCECs differentiation along several passages in hCECs co-cultured with either type of feeder layer. Therefore, the iHFL feeder layer appears to be the most effective at maintaining the proliferative properties of hCECs in culture most likely by preserving high levels of Sp1 and low levels of NFIB, which is known for its gene repressor and cell differentiation properties.
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Alcalde, Ignacio, Cristina Sánchez-Fernández, Susana Del Olmo-Aguado, Carla Martín, Céline Olmiere, Enol Artime, Luis M. Quirós e Jesús Merayo-Lloves. "Synthetic Heparan Sulfate Mimetic Polymer Enhances Corneal Nerve Regeneration and Wound Healing after Experimental Laser Ablation Injury in Mice". Polymers 14, n.º 22 (15 de novembro de 2022): 4921. http://dx.doi.org/10.3390/polym14224921.
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(1) Background: Abnormal corneal wound healing compromises visual acuity and can lead to neuropathic pain. Conventional treatments usually fail to restore the injured corneal tissue. In this study, we evaluated the effectiveness of a synthetic heparan sulfate mimetic polymer (HSmP) in a mouse model of corneal wound healing. (2) Methods: A surgical laser ablation affecting the central cornea and subbasal nerve plexus of mice was used as a model of the wound-healing assay. Topical treatment with HSmP was contrasted to its vehicle and a negative control (BSS). Corneal repair was studied using immunofluorescence to cell proliferation (Ki67), apoptosis (TUNEL assay), myofibroblast transformation (αSMA), assembly of epithelial cells (E-cadherin) and nerve regeneration (β-tubulin III). (3) Results: At the end of the treatment, normal epithelial cytoarchitecture and corneal thickness were achieved in HSmP-treated animals. HSmP treatment reduced myofibroblast occurrence compared to eyes irrigated with vehicle (p < 0.01) or BSS (p < 0.001). The HSmP group showed 50% more intraepithelial nerves than the BSS or vehicle groups. Only HSmP-treated corneas improved the visual quality to near transparent. (4) Conclusions: These results suggest that HSmP facilitates the regeneration of the corneal epithelium and innervation, as well as restoring transparency and reducing myofibroblast scarring after laser experimental injury.
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Nili, Elham, Fiona J. Li, Rebecca A. Dawson, Cora Lau, Blair McEwan, Nigel L. Barnett, Steven Weier, Jennifer Walshe, Neil A. Richardson e Damien G. Harkin. "The Impact of Limbal Mesenchymal Stromal Cells on Healing of Acute Ocular Surface Wounds Is Improved by Pre-cultivation and Implantation in the Presence of Limbal Epithelial Cells". Cell Transplantation 28, n.º 9-10 (17 de junho de 2019): 1257–70. http://dx.doi.org/10.1177/0963689719858577.
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While limbal epithelial cells are used for treating ocular surface wounds, the therapeutic potential of mesenchymal cells cultivated from the limbal stroma (LMSC) is less clear. We have therefore examined the effects of LMSC when applied to acute ocular surface wounds. LMSC derived from male rabbits (RLMSC) were applied to the ocular surface of female rabbits immediately following removal of the corneal and limbal epithelium. Human amniotic membrane (HAM) was used as the vehicle for implanting the RLMSC. The effects of RLMSC were examined when applied alone ( n = 3) and in conjunction with a stratified culture of human limbal epithelial cells (HLE) grown on the opposing surface of the HAM ( n = 3). Outcomes were monitored over 3 months in comparison with animals receiving no treatment ( n = 3) or treatment with HLE alone on HAM ( n = 3). Animals treated with RLMSC ( n = 6) displayed faster re-epithelialization (∼90% versus 70% healing after 12 weeks), with best results being observed when RLMSC were pre-cultivated and implanted in the presence of HLE ( p < 0.01; 90% healing by 7 weeks). While all animals displayed conjunctival cells on the corneal surface (by presence of goblet cells and/or keratin 13 expression) and corneal neovascularization, evidence of corneal epithelial regeneration was observed in animals that received RLMSC in the presence of HLE (by staining for keratin 3 and the absence of goblet cells). Conversely, corneal neovascularization was significantly greater when RLMSC were applied in the absence of HLE (<0.05; 90% of cornea compared with 20–30% in other cohorts). Nevertheless, neither human nuclear antigen nor rabbit Y chromosome were detected within the regenerated epithelium. Our results demonstrate that while cultured LMSC encourage corneal re-epithelialization, healing is improved by the pre-cultivation and implantation of these mesenchymal cells in the presence of limbal epithelial cells.