WorldWideScience

Sample records for corneal epithelial cells

  1. Trefoil peptides promote restitution of wounded corneal epithelial cells.

    Science.gov (United States)

    Göke, M N; Cook, J R; Kunert, K S; Fini, M E; Gipson, I K; Podolsky, D K

    2001-04-01

    The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.

  2. Oxidized alginate hydrogels as niche environments for corneal epithelial cells.

    Science.gov (United States)

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-10-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2-0.8 µm) than unmodified gels (pore diameter: 0.05-0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

  3. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    Science.gov (United States)

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  4. Limbal stem cells: Central concepts of corneal epithelial homeostasis

    Institute of Scientific and Technical Information of China (English)

    Jinny; J; Yoon; Salim; Ismail; Trevor; Sherwin

    2014-01-01

    A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface

  5. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

    Directory of Open Access Journals (Sweden)

    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  6. The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Ling Yu; Jian Ge; Zhichong Wang; Bing Huang; Keming Yu; Chongde Long; Xigu Chen

    2001-01-01

    Purpose:To study preliminarily induced differentiation of embryonic stem cells intocorneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal cornealepithelial cells in Transwell system to induce differentiation. Mophological andimmunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance.The cells formed a network and were confluent into film gradually after beingco-cultured with rabbit limbal corneal epithelial cells for 24 ~ 96 hours. The cells rangedmosaic structure and localized together with clear rim. Most of the cells showedpolygonal appearance. Transmission electron microscope showed lots of microvilli on thesurface of induced cells and tight junctions between them. These epithelial-like cellsexpressed the corneal epithelial cell specific marker cytokeratin3/cytokeratinl2.Conclusion: The potential mechanism of the differentiation of murine embryonic stemcells into corneal epithelial cells induced by limbal corneal epithelial cell-derivedinducing activity is to be further verified.

  7. Protective Effects of Trehalose on the Corneal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Pasquale Aragona

    2014-01-01

    Full Text Available Purpose. Aim of the present work was to evaluate the effects of the trehalose on the corneal epithelium undergoing alcohol delamination. Methods. Twelve patients undergoing laser subepithelial keratomileusis (LASEK were consecutively included in the study. The right eyes were pretreated with 3% trehalose eye drops, whilst left eyes were used as control. Epithelial specimens were processed for cells vitality assessment, apoptosis, and light and transmission electron microscopy; a morphometric analysis was performed in both groups. Results. In both trehalose-untreated eyes (TUE and trehalose-treated eyes (TTE, the percentage of vital cells was similar and no apoptotic cells were observed. In TUE, the corneal epithelium showed superficial cells with reduced microfolds, wing cells with vesicles and dilated intercellular spaces, and dark basal cells with vesicles and wide clefts. In TTE, superficial and wing cells were better preserved, and basal cells were generally clear with intracytoplasmatic vesicles. The morphometric analysis showed statistically significant differences between the two groups: the TTE epithelial height was higher, the basal cells showed larger area and clearer cytoplasm. The distribution of desmosomes and hemidesmosomes was significantly different between the groups. Conclusions. Trehalose administration better preserved morphological and morphometric features of alcohol-treated corneal epithelium, when compared to controls.

  8. Effects of N-acetylcysteine on matrix metalloproteinase-9 secretion and cell migration of human corneal epithelial cells

    OpenAIRE

    Ramaesh, T; Ramaesh, K; Riley, S C; West, J.D.; Dhillon, B

    2012-01-01

    Matrix metalloproteinase-9 (MMP-9) secreted by corneal epithelial cells has a role in the remodelling of extracellular matrix and migration of epithelial cells. Elevated levels of MMP-9 activity in the ocular surface may be involved in the pathogenesis of corneal diseases. N-acetylcysteine (NAC) has been used to treat corneal diseases, including recurrent epithelial erosions. In this study, its effects on the MMP-9 secretion and human corneal epithelial (HCE) cell migration were evaluated in ...

  9. The Effects of Vitamin A Compounds on Hyaluronic Acid Released from Cultured Rabbit Corneal Epithelial Cells and Keratocytes

    National Research Council Canada - National Science Library

    TOSHIDA, Hiroshi; TABUCHI, Nobuhito; KOIKE, Daisuke; KOIDE, Misao; SUGIYAMA, Keikichi; NAKAYASU, Kiyoo; KANAI, Atsushi; MURAKAMI, Akira

    2012-01-01

    .... Hyaluronic acid is produced by corneal epithelial cells and keratocytes in the eye. We investigated whether rabbit corneal epithelial cells and keratocytes release hyaluronic acid after exposure to vitamin A compounds...

  10. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    Science.gov (United States)

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  11. Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

    Science.gov (United States)

    Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A

    2014-06-01

    It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

  12. In vitro ultraviolet–induced damage in human corneal, lens, and retinal pigment epithelial cells

    OpenAIRE

    Youn, Hyun-Yi; McCanna, David J.; Sivak, Jacob G.; Jones, Lyndon W.

    2011-01-01

    Purpose The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Methods Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated w...

  13. A comparative evaluation of corneal epithelial cell cultures for assessing ocular permeability.

    Science.gov (United States)

    Becker, Ulrich; Ehrhardt, Carsten; Schneider, Marc; Muys, Leon; Gross, Dorothea; Eschmann, Klaus; Schaefer, Ulrich F; Lehr, Claus-Michael

    2008-02-01

    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.

  14. Nerve growth factor modulate proliferation of cultured rabbit corneal endothelial cells and epithelial cells.

    Science.gov (United States)

    Li, Xinyu; Li, Zhongguo; Qiu, Liangxiu; Zhao, Changsong; Hu, Zhulin

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  15. NK cells are necessary for recovery of corneal CD11c+ dendritic cells after epithelial abrasion injury

    Science.gov (United States)

    Mechanisms controlling CD11c(+) MHCII(+) DCs during corneal epithelial wound healing were investigated in a murine model of corneal abrasion. Selective depletion of NKp46(+) CD3- NK cells that normally migrate into the cornea after epithelial abrasion resulted in >85% reduction of the epithelial CD1...

  16. Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Aihua Hou

    Full Text Available PURPOSE: Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. METHODS: Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. RESULTS: Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. CONCLUSION: Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells.

  17. Identification of human fibroblast cell lines as a feeder layer for human corneal epithelial regeneration.

    Directory of Open Access Journals (Sweden)

    Rong Lu

    Full Text Available There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE and cell growth capacity were evaluated on days 5-14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1 × 10(4 in a 35-mm dish (9.6 cm(2 grew to confluence (about 1.87-2.41 × 10(6 cells in 12-14 days, representing 187-241 fold expansion with over 7-8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.

  18. The cytokine regulation of SPARC production by rabbit corneal epithelial cells and fibroblasts in vitro.

    Science.gov (United States)

    Abe, Kosuke; Hibino, Tsuyoshi; Mishima, Hiroshi; Shimomura, Yoshikazu

    2004-03-01

    SPARC (osteonectin/BM40) is detected in the corneal stroma during the wound-healing process. To understand the metabolism of SPARC in the cornea, we investigated the effects of cytokines and growth factors on SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. Rabbit corneal epithelial cells or fibroblasts were cultured for 3 days with serum-containing minimal essential medium (MEM), then subcultured for 3 days on serum-free MEM with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), or interleukin-1beta (IL-1beta). SPARC concentration in the medium was measured by the ELISA method using anti-SPARC monoclonal antibody. The concentration of SPARC in the conditioned medium of the epithelial cells depended on either cell numbers or cultivation periods. When EGF was added to the medium, the amount of SPARC in the medium decreased. The addition of IL-1beta, PDGF, or TGF-beta did not affect SPARC synthesis by the epithelial cells. The production of SPARC by rabbit corneal fibroblasts was low compared with that by epithelial cells. However, the synthesis of SPARC by corneal fibroblasts was significantly enhanced by the addition of TGF-beta. The addition of IL-1beta, PDGF, or EGF slightly increased SPARC synthesis by corneal fibroblasts. Cytokines and growth factors modulate SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. These results suggest that cytokines and growth factors modulate cell-matrix interaction in corneal wound healing, possibly by regulating SPARC synthesis.

  19. Limbal Stromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells.

    Science.gov (United States)

    Katikireddy, Kishore Reddy; Jurkunas, Ula V

    2016-01-01

    From the derivation of the first human embryonic stem (hES) cell line to the development of induced pluripotent stem (iPS) cells; it has become evident that tissue specific stem cells are able to differentiate into a specific somatic cell types. The understanding of key processes such as the signaling pathways and the role of the microenvironment in epidermal/epithelial development has provided important clues for the derivation of specific epithelial cell types.Various differentiation protocols/methods were used to attain specific epithelial cell types. Here, we describe in detail the procedure to follow for isolation of tissue specific stem cells, mimicking their microenvironment to attain stem cell characteristics, and their potential differentiation to corneal epithelial cells.

  20. Hyaluronate Acid-Dependent Protection and Enhanced Corneal Wound Healing against Oxidative Damage in Corneal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jing Zhong

    2016-01-01

    Full Text Available Purpose. To evaluate the effects and mechanism of exogenous hyaluronate (HA in promoting corneal wound healing. Methods. Human corneal epithelial cells (HCECs were incubated with different concentrations of HA to evaluate their efficiency in promoting cell migration and their modulation of repair factors. After inducing hyperosmolar conditions, the cell morphologies, cell apoptosis, and expression levels of TNF-α and MMP-9 were detected to assess the protective role of HA. Corneal epithelium-injured rat models were established to test the therapeutic effects of 0.3% HA. Then, the wound healing rates, the RNA expression levels of inflammatory cytokines, and repair factors were examined. Results. HCECs in the 0.03% and 0.3% HA groups showed fewer morphological alterations and lower rates of cell apoptosis following preincubation with HA under hyperosmolar conditions, as well as the expression levels of MMP-9 and TNF-α. In the rat model, the areas of fluorescein staining in the corneas of 0.3% HA group were significantly smaller than the control group. The expression levels of IL-1β and MMP-9 were decreased, while CD44 and FN were increased in the 0.3% HA group. Conclusion. HA enhanced corneal epithelial cell wound healing by promoting cell migration, upregulating repair responses, and suppressing inflammatory responses.

  1. Hyaluronate Acid-Dependent Protection and Enhanced Corneal Wound Healing against Oxidative Damage in Corneal Epithelial Cells.

    Science.gov (United States)

    Zhong, Jing; Deng, Yuqing; Tian, Bishan; Wang, Bowen; Sun, Yifang; Huang, Haixiang; Chen, Ling; Ling, Shiqi; Yuan, Jin

    2016-01-01

    Purpose. To evaluate the effects and mechanism of exogenous hyaluronate (HA) in promoting corneal wound healing. Methods. Human corneal epithelial cells (HCECs) were incubated with different concentrations of HA to evaluate their efficiency in promoting cell migration and their modulation of repair factors. After inducing hyperosmolar conditions, the cell morphologies, cell apoptosis, and expression levels of TNF-α and MMP-9 were detected to assess the protective role of HA. Corneal epithelium-injured rat models were established to test the therapeutic effects of 0.3% HA. Then, the wound healing rates, the RNA expression levels of inflammatory cytokines, and repair factors were examined. Results. HCECs in the 0.03% and 0.3% HA groups showed fewer morphological alterations and lower rates of cell apoptosis following preincubation with HA under hyperosmolar conditions, as well as the expression levels of MMP-9 and TNF-α. In the rat model, the areas of fluorescein staining in the corneas of 0.3% HA group were significantly smaller than the control group. The expression levels of IL-1β and MMP-9 were decreased, while CD44 and FN were increased in the 0.3% HA group. Conclusion. HA enhanced corneal epithelial cell wound healing by promoting cell migration, upregulating repair responses, and suppressing inflammatory responses.

  2. The Toxicity of Nonsteroidal Anti-inflammatory Eye Drops against Human Corneal Epithelial Cells in Vitro.

    Science.gov (United States)

    Lee, Jong Soo; Kim, Young Hi; Park, Young Min

    2015-12-01

    This study investigated the toxicity of commercial non-steroid anti-inflammatory drug (NSAID) eye solutions against corneal epithelial cells in vitro. The biologic effects of 1/100-, 1/50-, and 1/10-diluted bromfenac sodium, pranoprofen, diclofenac sodium, and the fluorometholone on corneal epithelial cells were evaluated after 1-, 4-, 12-, and 24-hr of exposure compared to corneal epithelial cell treated with balanced salt solution as control. Cellular metabolic activity, cellular damage, and morphology were assessed. Corneal epithelial cell migration was quantified by the scratch-wound assay. Compared to bromfenac and pranoprofen, the cellular metabolic activity of diclofenac and fluorometholone significantly decreased after 12-hr exposure, which was maintained for 24-hr compared to control. Especially, at 1/10-diluted eye solution for 24-hr exposure, the LDH titers of fluorometholone and diclofenac sodium markedly increased more than those of bromfenac and pranoprofen. In diclofenac sodium, the Na(+) concentration was lower and amount of preservatives was higher than other NSAIDs eye solutions tested. However, the K(+) and Cl(-) concentration, pH, and osmolarity were similar for all NSAIDs eye solutions. Bromfenac and pranoprofen significantly promoted cell migration, and restored wound gap after 48-hr exposure, compared with that of diclofenac or fluorometholone. At 1/50-diluted eye solution for 48-hr exposure, the corneal epithelial cellular morphology of diclofenac and fluorometholone induced more damage than that of bromfenac or pranoprofen. Overall, the corneal epithelial cells in bromfenac and pranoprofen NSAID eye solutions are less damaged compared to those in diclofenac, included fluorometholone as steroid eye solution.

  3. Experimental Study of Plasmid TGF-β1 DNA Gene Transfer with Lipofectamine into Rabbit Corneal Epithelial Cells In Vitro

    Institute of Scientific and Technical Information of China (English)

    黄琼; 胡燕华; 姜发纲; 陈宏

    2002-01-01

    To investigate whether the TGF-β1 plasmid DNA carried by lipofectamine could be introduced into cultured rabbit corneal epithelial cells, specific expression of the plasmid pMAM TGF-β1in the cultured corneal epithelial cells was studied. Two days after 12 h of transfection of pMAMTGF-β1 mediated by lipofectamine into the cultured corneal epithelial cells, the TGF-β1 protein expression specific for pMAMTGF-β1 in the cells was detected by means of immunohistochemical staining and the positive rate was 23. 37 %. The results suggested that foreign plasmid DNA could be effectively delivered into cultured rabbit corneal epithelial cells by means of lipofectamine, and this will provide a promising method of studying TGF-β1 on the mechanism of physiology and pathology concerned with corneal epithelial cells.

  4. ICAM-1 is necessary for epithelial recruitment of gammadelta T cells and efficient corneal wound healing.

    Science.gov (United States)

    Wound healing and inflammation are both significantly reduced in mice that lack gammadelta T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in gammadelta T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epi...

  5. NK cells modulate the inflammatory response to corneal epithelial abrasion and thereby support wound healing

    Science.gov (United States)

    Natural killer cells are lymphocytes of the innate immune system that have crucial cytotoxic and regulatory roles in adaptive immunity and inflammation. Herein, we consider a role for these cells in corneal wound healing. After a 2-mm central epithelial abrasion of the mouse cornea, a subset of clas...

  6. Rebamipide increases the mucin-like glycoprotein production in corneal epithelial cells.

    Science.gov (United States)

    Takeji, Yasuhiro; Urashima, Hiroki; Aoki, Akihiro; Shinohara, Hisashi

    2012-06-01

    Dry eye is a multifactorial disease of tears and the ocular surface due to tear deficiency or excessive tear evaporation. Tear film instability is due to a disturbance in ocular surface mucin leading to a dysfunction of mucin, resulting in dry eye. In this study, we examined the effect of rebamipide, an anti-ulcer agent, on glycoconjugate production, as an indicator of mucin-like glycoprotein in cultured corneal epithelial cells. Further, we investigated the effect of rebamipide on the gene expression of membrane-associated mucins. Confluent cultured human corneal epithelial cells were incubated with rebamipide for 24 h. The glycoconjugate content in the supernatant and the cell extracts was measured by wheat germ agglutinin-enzyme-linked lectin assay combined gel-filtration method. In the experiment on mucin gene expression, cultured human corneal epithelial cells were collected at 0, 3, 6, and 12 h after administration of rebamipide. Real-time quantitative polymerase chain reaction was used to analyze the quantity of MUC1, MUC 4, and MUC16 gene expression. Rebamipide significantly increased the glycoconjugate contents in the supernatant and cell extract. In the mucin gene expression in the cells, rebamipide increased MUC1 and MUC4 gene expression, but did not increase MUC16 gene expression. Rebamipide promoted glycoconjugate, which has a property as a mucin-like glycoprotein, in human corneal epithelial cells. The increased production was mediated by MUC1 and MUC4 gene expression.

  7. In Vitro Effects of Preserved and Unpreserved Anti-Allergic Drugs on Human Corneal Epithelial Cells

    OpenAIRE

    Guzman-Aranguez, Ana; Calvo, Patricia; Ropero, Inés; Pintor, Jesús

    2014-01-01

    Purpose: Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells.

  8. Efficacy of cultivated corneal epithelial stem cells for ocular surface reconstruction

    Directory of Open Access Journals (Sweden)

    Prabhasawat P

    2012-09-01

    Full Text Available Pinnita Prabhasawat,1 Pattama Ekpo,2 Mongkol Uiprasertkul,3 Suksri Chotikavanich,1 Nattaporn Tesavibul11Department of Ophthalmology, 2Department of Immunology, 3Department of Pathology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, ThailandPurpose: To investigate the clinical outcomes of cultivated corneal limbal epithelial transplantation (CLET using human amniotic membrane for corneal limbal stem-cell deficiency.Methods: Prospective, noncomparative case series. Eighteen patients (19 eyes with severe ocular surface diseases were chosen to undergo CLET using human amniotic membrane. Twelve eyes received auto-CLET, and seven eyes received allo-CLET. Clinical outcomes of corneal surface epithelialization, conjunctivalization, inflammation, visual acuity, graft status, and complications were observed.Results: Corneal epithelium cultivated on amniotic membrane (two to four layers was positive for molecular markers p63, ABCG2, CK3, and CK12. The mean patient age was 44.7 ± 15.2 years. A successful clinical outcome, defined as corneal epithelialization without central conjunctivalization or severe inflammation, was obtained in 14 (73.7% of 19 eyes (mean follow-up 26.1 ± 13.5 months; range 6–47. A histopathologic success, defined as absence of goblet cells at the central cornea, was achieved in 12 (63.2% eyes. Clinical failures occurred in five (26.3% of 19 eyes, and histopathologic failures occurred in seven (36.8% of 19 eyes. Survival analysis at 1 year showed that the clinical success rate was 77.9% and the pathological success rate was 72.3%. Fourteen of 19 (73.7% eyes had visual acuity improvements after CLET. Six cases underwent penetrating keratoplasty; five of these grafts remained clear after 20.4 ± 6.9 months (range, 12–31 of follow-up. Complications included infectious keratitis (three cases and recurrent symblepharon (one case. All complicated cases had lid abnormalities. Factors affecting the final clinical

  9. Roles of TRIM32 in Corneal Epithelial Cells After Infection with Herpes Simplex Virus

    Directory of Open Access Journals (Sweden)

    Hao Cui

    2017-09-01

    Full Text Available 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.

  10. Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction.

    Science.gov (United States)

    Mikhailova, Alexandra; Ilmarinen, Tanja; Ratnayake, Anjula; Petrovski, Goran; Uusitalo, Hannu; Skottman, Heli; Rafat, Mehrdad

    2016-05-01

    Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD) - a painful and vision-threatening condition. Treating these disorders is often challenging and complex, especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSC-derived LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation of hPSCs towards LESC-like cells was directed using small-molecule induction followed by maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness, using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15, along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a novel tissue engineering approach for

  11. A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology.

    Science.gov (United States)

    Shaharuddin, Bakiah; Ahmad, Sajjad; Md Latar, Nani; Ali, Simi; Meeson, Annette

    2016-10-14

    : Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Limbal stem cell deficiency is a painful eye condition caused by abnormal maintenance of limbal stem cells. It is treated by transplantation of limbal epithelial cells derived from human tissue. The success of this treatment depends of the quality of the cells transplanted; however, some transplants fail. Understanding more about the immunobiology of these cells within the transplants could

  12. The effect of Lamium album extract on cultivated human corneal epithelial cells (10.014 pRSV-T

    Directory of Open Access Journals (Sweden)

    Roman Paduch

    2015-01-01

    Conclusion: Selected Lamium album extracts influence human corneal epithelial cells. Generally, while not toxic, they modulate pro-inflammatory and anti-inflammatory cytokines levels, and decrease NO release by cells; moreover, ethanol and ethyl acetate extracts reduce ROS levels.

  13. Ascorbic Acid Promotes the Stemness of Corneal Epithelial Stem/Progenitor Cells and Accelerates Epithelial Wound Healing in the Cornea.

    Science.gov (United States)

    Chen, Jialin; Lan, Jie; Liu, Dongle; Backman, Ludvig J; Zhang, Wei; Zhou, Qingjun; Danielson, Patrik

    2017-03-09

    High concentration of ascorbic acid (vitamin C) has been found in corneal epithelium of various species. However, the specific functions and mechanisms of ascorbic acid in the repair of corneal epithelium are not clear. In this study, it was found that ascorbic acid accelerates corneal epithelial wound healing in vivo in mouse. In addition, ascorbic acid enhanced the stemness of cultured mouse corneal epithelial stem/progenitor cells (TKE2) in vitro, as shown by elevated clone formation ability and increased expression of stemness markers (especially p63 and SOX2). The contribution of ascorbic acid on the stemness enhancement was not dependent on the promotion of Akt phosphorylation, as concluded by using Akt inhibitor, nor was the stemness found to be dependent on the regulation of oxidative stress, as seen by the use of two other antioxidants (GMEE and NAC). However, ascorbic acid was found to promote extracellular matrix (ECM) production, and by using two collagen synthesis inhibitors (AzC and CIS), the increased expression of p63 and SOX2 by ascorbic acid was decreased by around 50%, showing that the increased stemness by ascorbic acid can be attributed to its regulation of ECM components. Moreover, the expression of p63 and SOX2 was elevated when TKE2 cells were cultured on collagen I coated plates, a situation that mimics the in vivo situation as collagen I is the main component in the corneal stroma. This study shows direct therapeutic benefits of ascorbic acid on corneal epithelial wound healing and provides new insights into the mechanisms involved. © Stem Cells Translational Medicine 2017.

  14. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    Directory of Open Access Journals (Sweden)

    Chi-Chin Sun

    Full Text Available Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

  15. Induction of corneal epithelial progenitors from bone-marrow mesenchymal stem cells of rhesus monkeys in vitro

    Institute of Scientific and Technical Information of China (English)

    YUAN Jing; YU JianXiong; HUANG Bing; LIU BingQian; LIU JingBo; JIANG RuZhang; GE Jian

    2007-01-01

    Bioengineered corneas are substitutes for human donor tissue that are designed to treat severe disease affecting ocular surfaces.However, a shortage of candidate seed cells for bioengineering corneas is still a problem.Bone-marrow mesenchymal stem cells (MSCs) are capable of multilineage differentiation.Therefore, we determined whether MSCs differentiate into corneal epithelial cells (ECs).We applied three exoteric-microenvironmental systems to induce MSCs to become ECs.Induced MSC were identified by means of morphologic examination, immunocytochemical analysis, and flow cytometry.MSCs grown in one microenvironment had characteristics similar to those of corneal epithelial progenitors.Induced MSCs expressed markers for EC, including integrin β1, Cx43, Pax6, and P63.MSCs were successfully induced to become corneal epithelial progenitors.Therefore, the use of MSCs may hold substantial promise for reconstructing the ocular surface after corneal injury.

  16. The SULFs, extracellular sulfatases for heparan sulfate, promote the migration of corneal epithelial cells during wound repair.

    Directory of Open Access Journals (Sweden)

    Inna Maltseva

    Full Text Available Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS. SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1⁻/⁻, but not Sulf2⁻/⁻, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1⁻/⁻ mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE. Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.

  17. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xin, Ying [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Lu, Qingxian [Tumor Immunobiology Group, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Li, Qiutang, E-mail: q.li@louisville.edu [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States)

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  18. Differential expression of epithelial basement membrane components nidogens and perlecan in corneal stromal cells in vitro.

    Science.gov (United States)

    Santhanam, Abirami; Torricelli, Andre A M; Wu, Jiahui; Marino, Gustavo K; Wilson, Steven E

    2015-01-01

    The purpose of this study was to examine the expression of corneal epithelial basement membrane (EBM) components in different corneal stromal cell types. In vitro model systems were used to explore the expression of EBM components nidogen-1, nidogen-2, and perlecan that are the primary components in the lamina lucida and the lamina densa that defectively regenerate in corneas with stromal opacity after in -9.0 D photorefractive keratectomy (PRK). Primary rabbit corneal stromal cells were cultured using varying serum concentrations and exogenous growth factors, including fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-β1, to optimize the growth of each cell type of interest. The expression of the keratocyte-specific marker keratocan and the myofibroblast-specific marker α-smooth muscle actin (α-SMA) were analyzed with real-time PCR, western blot, and immunocytochemical staining to evaluate the specificity of the cell types and select optimal conditions (high keratocan and low α-SMA for keratocytes; low keratocan and high α-SMA for myofibroblasts; low keratocan and low α-SMA for corneal fibroblasts). The expression of the EBM components nidogen-1, nidogen-2, and perlecan was evaluated in each corneal cell type using real-time PCR, immunostaining, and western blotting. In agreement with previous studies, serum-free DMEM was found to be optimal for keratocytes, DMEM with 10% serum and 40 ng/ml FGF-2 yielded the best marker profile for corneal fibroblasts, and DMEM with 1% serum and 2 ng/ml TGF-β1 was found to be optimal for myofibroblasts. Nidogen-1 and nidogen-2 mRNAs were highly expressed in keratocytes, whereas perlecan was highly expressed in myofibroblasts. In keratocytes, nidogen-2 and perlecan proteins were expressed predominantly in intracellular compartments, whereas in myofibroblasts expression of both EBM components was observed diffusely throughout the cell. Although the perlecan mRNA levels were high in the myofibroblasts, the

  19. Expression of Phospholipases A2 and C in Human Corneal Epithelial Cells

    Science.gov (United States)

    Landreville, Solange; Coulombe, Stéphanie; Carrier, Patrick; Gelb, Michael H.; Guérin, Sylvain L.; Salesse, Christian

    2008-01-01

    Purpose To achieve a better understanding of the involvement of phospholipases in the inflammation and wound-healing processes in human corneal epithelial cells (HCECs), expression of phospholipase A2s (PLA2s) and phospholipase Cs (PLCs) was examined in the human corneal epithelium. Methods Specific primers were designed for RT-PCR amplification of the known secreted (s)PLA2, cytosolic (c)PLA2, and PLC mRNAs. Corresponding PCR products were cloned and the DNA sequenced. Immunofluorescence of flatmounted corneal sections and Western blot analyses were used to detect the PLA2s and PLCs expressed by HCECs. Results The mRNAs for the following phospholipases were detected by RT-PCR in the HCECs: sPLA2GIII, -GX, and -GXIIA; cPLA2α and -γ; PLCβ1, -β2, -β3, -β4, -γ1, -γ2, -δ1, -δ3, -δ4, and -ε. Immunofluorescence analyses conducted on corneal epithelium cryosections and Western blot on freshly isolated HCECs demonstrated the presence of sPLA2GIII, -GX, and -GXIIA; cPLA2α and -γ; and PLCβ2, -β3, -γ1, -γ2, and -δ3. Conclusions Many phospholipase isoforms are expressed by HCECs and may play a major role in signal transduction (PLCs) as well as in the release of precursors of potent mediators of inflammation, such as leukotrienes and prostaglandins (PLA2s). Moreover, the sPLA2s expressed by the corneal epithelium could be involved in the normal antibacterial activity in the tears and in wound healing. PMID:15505048

  20. Protection of human corneal epithelial cells from TNF-α-induced disruption of barrier function by rebamipide.

    Science.gov (United States)

    Kimura, Kazuhiro; Morita, Yukiko; Orita, Tomoko; Haruta, Junpei; Takeji, Yasuhiro; Sonoda, Koh-Hei

    2013-04-17

    TNF-α disrupts the barrier function of cultured human corneal epithelial (HCE) cells. We investigated the effects of the cytoprotective drug rebamipide on this barrier disruption by TNF-α as well as on corneal epithelial damage in a rat model of dry eye. The barrier function of HCE cells was evaluated by measurement of transepithelial electrical resistance. The distribution of tight-junction (ZO-1, occludin) and adherens-junction (E-cadherin, β-catenin) proteins, and the p65 subunit of nuclear factor-κB (NF-κB) was determined by immunofluorescence microscopy. Expression of junctional proteins as well as phosphorylation of the NF-κB inhibitor IκB-α and myosin light chain (MLC) were examined by immunoblot analysis. A rat model of dry eye was developed by surgical removal of exorbital lacrimal glands. Rebamipide inhibited the disruption of barrier function as well as the downregulation of ZO-1 expression, and the disappearance of ZO-1 from the interfaces of neighboring HCE cells induced by TNF-α. It also inhibited the phosphorylation and downregulation of IκB-α, the translocation of p65 to the nucleus, the formation of actin stress fibers, and the phosphorylation of MLC induced by TNF-α in HCE cells. Treatment with rebamipide eyedrops promoted the healing of corneal epithelial defects as well as attenuated the loss of ZO-1 from the surface of corneal epithelial cells in rats. Rebamipide protects corneal epithelial cells from the TNF-α-induced disruption of barrier function by maintaining the distribution and expression of ZO-1 as well as the organization of the actin cytoskeleton. Rebamipide is, thus, a potential drug for preventing or ameliorating the loss of corneal epithelial barrier function associated with ocular inflammation.

  1. Human corneal epithelial subpopulations

    DEFF Research Database (Denmark)

    Søndergaard, Chris Bath

    2013-01-01

    -free EpiLife medium, using a range of physiologically relevant oxygen concentrations (2%, 5%, 10%, 15% and 20%). Using immunocytochemistry and advanced fluorescence microscopy, cells were characterized regarding growth, cell cycle distribution, colony-forming efficiency (CFE), phenotypes...... and cytomorphometry. Limbal epithelial cells expanded in 2% O2 exhibited slow growth, low fraction of cells in S/G2 , high CFE, high expression of stem cell markers ABCG2 and p63α, and low fraction of differentiation marker CK3 resembling a LESC phenotype. The effect of hypoxia to maintain LESCs in culture...

  2. The preservative polyquaternium-1 increases cytoxicity and NF-kappaB linked inflammation in human corneal epithelial cells

    OpenAIRE

    Paimela, Tuomas; Ryhänen, Tuomas; Kauppinen, Anu; Marttila, Liisa; Salminen, Antero; Kaarniranta, Kai

    2012-01-01

    Purpose In numerous clinical and experimental studies, preservatives present in eye drops have had detrimental effects on ocular epithelial cells. The aim of this study was to compare the cytotoxic and inflammatory effects of the preservative polyquaternium-1 (PQ-1) containing Travatan (travoprost 0.004%) and Systane Ultra eye drops with benzalkonium chloride (BAK) alone or BAK-preserved Xalatan (0.005% latanoprost) eye drops in HCE-2 human corneal epithelial cell culture. Methods HCE-2 cells...

  3. Fibroblast growth factor receptor 2 (FGFR2) is required for corneal epithelial cell proliferation and differentiation during embryonic development.

    Science.gov (United States)

    Zhang, Jinglin; Upadhya, Dinesh; Lu, Lin; Reneker, Lixing W

    2015-01-01

    Fibroblast growth factors (FGFs) play important roles in many aspects of embryonic development. During eye development, the lens and corneal epithelium are derived from the same surface ectodermal tissue. FGF receptor (FGFR)-signaling is essential for lens cell differentiation and survival, but its role in corneal development has not been fully investigated. In this study, we examined the corneal defects in Fgfr2 conditional knockout mice in which Cre expression is activated at lens induction stage by Pax6 P0 promoter. The cornea in LeCre, Fgfr2(loxP/loxP) mice (referred as Fgfr2(CKO)) was analyzed to assess changes in cell proliferation, differentiation and survival. We found that Fgfr2(CKO) cornea was much thinner in epithelial and stromal layer when compared to WT cornea. At embryonic day 12.5-13.5 (E12.5-13.5) shortly after the lens vesicle detaches from the overlying surface ectoderm, cell proliferation (judged by labeling indices of Ki-67, BrdU and phospho-histone H3) was significantly reduced in corneal epithelium in Fgfr2(CKO) mice. At later stage, cell differentiation markers for corneal epithelium and underlying stromal mesenchyme, keratin-12 and keratocan respectively, were not expressed in Fgfr2(CKO) cornea. Furthermore, Pax6, a transcription factor essential for eye development, was not present in the Fgfr2(CKO) mutant corneal epithelial at E16.5 but was expressed normally at E12.5, suggesting that FGFR2-signaling is required for maintaining Pax6 expression in this tissue. Interestingly, the role of FGFR2 in corneal epithelial development is independent of ERK1/2-signaling. In contrast to the lens, FGFR2 is not required for cell survival in cornea. This study demonstrates for the first time that FGFR2 plays an essential role in controlling cell proliferation and differentiation, and maintaining Pax6 levels in corneal epithelium via ERK-independent pathways during embryonic development.

  4. Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.

    Science.gov (United States)

    Chen, Jian; Guerriero, Emily; Lathrop, Kira; SundarRaj, Nirmala

    2008-01-01

    The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression. Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium. The number of cells in the S phase, the relative levels of specific cyclins and CDKs and their intracellular distribution, and the relative levels of mRNAs were determined by BrdU labeling, Western blot and immunocytochemical analyses, and real-time RT-PCR, respectively. ROCK inhibition delayed the progression of G(1) to S phase and led to a decrease in the number of RCECs entering the S phase between 12 and 24 hours from 31.5% +/- 4.5% to 8.1% +/- 2.6%. During the cell cycle progression, protein and mRNA levels of cyclin-D1 and -D3 and cyclin-dependent kinases CDK4 and CDK6 were significantly lower, whereas the protein levels of the CDK inhibitor p27(Kip1) were higher in ROCK-inhibited cells. Intracellular mRNA or protein levels of cyclin-E and protein levels of CDK2 were not significantly affected, but their nuclear translocation was delayed by ROCK inhibition. ROCK signaling is involved in cell cycle progression in RCECs, possibly by upregulation of cyclin-D1 and -D3 and CDK4, -6, and -2; nuclear translocation of CDK2 and cyclin-E; and downregulation of p27(Kip1).

  5. Influence of extracellular matrix proteins and substratum topography on corneal epithelial cell alignment and migration.

    Science.gov (United States)

    Raghunathan, Vijaykrishna; McKee, Clayton; Cheung, Wai; Naik, Rachel; Nealey, Paul F; Russell, Paul; Murphy, Christopher J

    2013-08-01

    The basement membrane (BM) of the corneal epithelium presents biophysical cues in the form of topography and compliance that can impact the phenotype and behaviors of cells and their nuclei through modulation of cytoskeletal dynamics. In addition, it is also well known that the intrinsic biochemical attributes of BMs can modulate cell behaviors. In this study, the influence of the combination of exogenous coating of extracellular matrix proteins (ECM) (fibronectin-collagen [FNC]) with substratum topography was investigated on cytoskeletal architecture as well as alignment and migration of immortalized corneal epithelial cells. In the absence of FNC coating, a significantly greater percentage of cells aligned parallel with the long axis of the underlying anisotropically ordered topographic features; however, their ability to migrate was impaired. Additionally, changes in the surface area, elongation, and orientation of cytoskeletal elements were differentially influenced by the presence or absence of FNC. These results suggest that the effects of topographic cues on cells are modulated by the presence of surface-associated ECM proteins. These findings have relevance to experiments using cell cultureware with biomimetic biophysical attributes as well as the integration of biophysical cues in tissue-engineering strategies and the development of improved prosthetics.

  6. Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

    Science.gov (United States)

    Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

    2007-01-01

    Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

  7. Biomimetic stochastic topography and electric fields synergistically enhance directional migration of corneal epithelial cells in a MMP-3-dependent manner.

    Science.gov (United States)

    Gao, Jing; Raghunathan, Vijay Krishna; Reid, Brian; Wei, Dongguang; Diaz, Rodney C; Russell, Paul; Murphy, Christopher J; Zhao, Min

    2015-01-01

    Directed migration of corneal epithelial cells (CECs) is critical for maintenance of corneal homeostasis as well as wound healing. Soluble cytoactive factors and the intrinsic chemical attributes of the underlying extracellular matrix (ECM) participate in stimulating and directing migration. The central importance of the intrinsic biophysical attributes of the microenvironment of the cell in modulating an array of fundamental epithelial behaviors including migration has been widely documented. Among the best measures of these attributes are the intrinsic topography and stiffness of the ECM and electric fields (EFs). How cells integrate these multiple simultaneous inputs is not well understood. Here, we present a method that combines the use of (i) topographically patterned substrates (mean pore diameter 800nm) possessing features that approximate those found in the native corneal basement membrane; and (ii) EFs (0-150mVmm(-1)) mimicking those at corneal epithelial wounds that the cells experience in vivo. We found that topographic cues and EFs synergistically regulated directional migration of human CECs and that this was associated with upregulation of matrix metalloproteinase-3 (MMP3). MMP3 expression and activity were significantly elevated with 150mVmm(-1) applied-EF while MMP2/9 remained unaltered. MMP3 expression was elevated in cells cultured on patterned surfaces against planar surfaces. The highest single-cell migration rate was observed with 150mVmm(-1) applied EF on patterned and planar surfaces. When cultured as a confluent sheet, EFs induced collective cell migration on stochastically patterned surfaces compared with dissociated single-cell migration on planar surfaces. These results suggest significant interaction of biophysical cues in regulating cell behaviors and will help define design parameters for corneal prosthetics and help to better understand corneal wound healing.

  8. Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell

    Directory of Open Access Journals (Sweden)

    Jing Dan

    2017-03-01

    Full Text Available AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2, transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in

  9. System for tracking transplanted limbal epithelial stem cells in the treatment of corneal stem cell deficiency

    Science.gov (United States)

    Boadi, J.; Sangwal, V.; MacNeil, S.; Matcher, S. J.

    2015-03-01

    The prevailing hypothesis for the existence and healing of the avascular corneal epithelium is that this layer of cells is continually produced by stem cells in the limbus and transported onto the cornea to mature into corneal epithelium. Limbal Stem Cell Deficiency (LSCD), in which the stem cell population is depleted, can lead to blindness. LSCD can be caused by chemical and thermal burns to the eye. A popular treatment, especially in emerging economies such as India, is the transplantation of limbal stem cells onto damaged limbus with hope of repopulating the region. Hence regenerating the corneal epithelium. In order to gain insights into the success rates of this treatment, new imaging technologies are needed in order to track the transplanted cells. Optical Coherence Tomography (OCT) is well known for its high resolution in vivo images of the retina. A custom OCT system has been built to image the corneal surface, to investigate the fate of transplanted limbal stem cells. We evaluate two methods to label and track transplanted cells: melanin labelling and magneto-labelling. To evaluate melanin labelling, stem cells are loaded with melanin and then transplanted onto a rabbit cornea denuded of its epithelium. The melanin displays strongly enhanced backscatter relative to normal cells. To evaluate magneto-labelling the stem cells are loaded with magnetic nanoparticles (20-30nm in size) and then imaged with a custom-built, magneto-motive OCT system.

  10. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

    Science.gov (United States)

    Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

    2015-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

  11. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

    Directory of Open Access Journals (Sweden)

    Chang Rae Rho

    Full Text Available Granulocyte-macrophage colony-stimulating factor (GM-CSF is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs. We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF. An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml. MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

  12. Phage display against corneal epithelial cells produced bioactive peptides that inhibit Aspergillus adhesion to the corneas.

    Directory of Open Access Journals (Sweden)

    Ge Zhao

    Full Text Available Dissection of host-pathogen interactions is important for both understanding the pathogenesis of infectious diseases and developing therapeutics for the infectious diseases like various infectious keratitis. To enhance the knowledge about pathogenesis infectious keratitis, a random 12-mer peptide phage display library was screened against cultured human corneal epithelial cells (HCEC. Fourteen sequences were obtained and BLASTp analysis showed that most of their homologue counterparts in GenBank were for defined or putative proteins in various pathogens. Based on known or predicted functions of the homologue proteins, ten synthetic peptides (Pc-A to Pc-J were measured for their affinity to bind cells and their potential efficacy to interfere with pathogen adhesion to the cells. Besides binding to HCEC, most of them also bound to human corneal stromal cells and umbilical endothelial cells to different extents. When added to HCEC culture, the peptides induced expression of MyD88 and IL-17 in HCEC, and the stimulated cell culture medium showed fungicidal potency to various extents. While peptides Pc-C and Pc-E inhibited Aspergillus fumigatus (A.f adhesion to HCEC in a dose-dependent manner, the similar inhibition ability of peptides Pc-A and Pc-B required presence of their homologue ligand Alb1p on A.f. When utilized in an eyeball organ culture model and an in vivo A.f keratitis model established in mouse, Pc-C and Pc-E inhibited fungal adhesion to corneas, hence decreased corneal disruption caused by inflammatory infiltration. Affinity pull-down of HCEC membrane proteins with peptide Pc-C revealed several molecules as potential receptors for this peptide. In conclusion, besides proving that phage display-selected peptides could be utilized to interfere with adhesion of pathogens to host cells, hence could be exploited for managing infectious diseases including infectious keratitis, we also proposed that the phage display technique and the

  13. Impact of Mycotoxins Secreted by Aspergillus Molds on the Inflammatory Response of Human Corneal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yélian Marc Bossou

    2017-06-01

    Full Text Available Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of aflatoxin B1 (AFB1 and gliotoxin, two mycotoxins secreted by Aspergillus molds, on the biological activity of the human corneal epithelial (HCE cells. After 24, 48, and 72 h of exposure, cellular viability and inflammatory response were assessed. Both endpoint cell viability colorimetric assays and continuous cell impedance measurements, providing noninvasive real-time assessment of the effect on cells, were performed. Cytokine gene expression and interleukin-8 release were quantified. Gliotoxin appeared more cytotoxic than AFB1 but, at the same time, led to a lower increase of the inflammatory response reflecting its immunosuppressive properties. Real-time cell impedance measurement showed a distinct profile of cytotoxicity for both mycotoxins. HCE cells appeared to be a well-suited in vitro model to study ocular surface reactivity following biological contaminant exposure. Low, but persistent inflammation, caused by environmental factors, such as fungal toxins, leads to irritation and sensitization, and could be responsible for allergic manifestations which, in turn, could lead to mucosal hyper-reactivity.

  14. Effects of biophysical and biochemical cues on human corneal epithelial cell behavior

    Science.gov (United States)

    Tocce, Elizabeth J.

    2011-12-01

    Recent advances in the design of biomaterials aim at mimicking the natural biophysical and biochemical components found in a tissue's extracellular environment (ECM). Of particular interest in this work is mimicking the specialized ECM of the human corneal epithelium called the basement membrane (BM) and understanding how corneal epithelial cells (HCECs) respond to biophysical and biochemical cues. To this end, well defined topographic features with dimension of the BM (20 to 200 nm) were fabricated to support controlled cell interactions with biochemical motifs (e.g., adhesive peptide ligands) found in the BM. Here, features of 30 to 70 nm that represent the smallest features found in the BM were used to demonstrate that the smallest features that HCECs can recognize are 30 and 45 nm, depending on the soluble environment. In addition, HCECs demonstrate contact guidance on the smallest BM features (30 to 70 nm) and on the largest BM features (200 nm), but differs from contact guidance on micron-scale features, suggesting that BM scale topography scale is an influential factor in regulating HCEC behavior. To study the simultaneous presentation of biophysical and biochemical cues, topographic features are coated with thin films using a layer-by-layer deposition of covalently reacting polymers, poly(ethylene imine) and poly(2-vinyl-4,4-dimethylazlactone (PEI/PVDMA). The films are functionalized with the bioactive peptide argenine-glycine-aspartic acid (RGD) to control cell-substrate interactions. We demonstrate that PEI/PVDMA films can be functionalized with monotonically increasing densities of ROD to control HCEC attachment and proliferation. In addition PEI/PVDMA films functionalized with RGD were used to demonstrate that HCEC response to topographic cues is dependent on the scale of the topography, the surface chemical composition and the soluble environment. Results from these studies will advance the understanding of how BM-relevant biophysical and biochemical

  15. Comparison of cytotoxicity and wound healing effect of carboxymethylcellulose and hyaluronic acid on human corneal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Jong; Soo; Lee; Seung; Uk; Lee; Cheng-Ye; Che; Ji-Eun; Lee

    2015-01-01

    AIM: To investigate the cytotoxic effect on human corneal epithelial cells(HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose(CMC) and hyaluronic acid(HA).METHODS: HCECs were exposed to 0.5% CMC(Refresh plus, Allergan, Irvine, California, USA) and 0.1% and 0.3%HA(Kynex , Alcon, Seoul, Korea, and Hyalein mini,Santen, Osaka, Japan) for the period of 30 min, and 4, 12,and 24 h. Methyl thiazolyl tetrazoiun(MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase(LDH) leakage assay to assess the cytotoxicity. apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24 h after confluent HCECs were scratch wounded.RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells weredemonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC.CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.

  16. The effect of K(+) on caspase activity of corneal epithelial cells exposed to UVB.

    Science.gov (United States)

    Leerar, John R; Glupker, Courtney D; Schotanus, Mark P; Ubels, John L

    2016-10-01

    Exposure of human corneal limbal epithelial (HCLE) cells to UVB triggers rapid loss of K(+) and apoptosis via activation of caspases -9, -8 and -3. It has been shown that preventing loss of intracellular K(+) can inhibit apoptosis. The goal of this study was to investigate the effect of K(+) on the UVB-induced caspase activity. HCLE cells were exposed to 150 mJ/cm(2) UVB, followed by measurement of caspase activity in cell lysates. Caspase activity was measured in the presence and absence of 100 mM K(+) in the reaction buffer. UVB-induced activity of caspases -9, -8 and -3 all decreased in the presence of 100 mM K(+). These results suggest that a role of high [K(+)] in the cell is to inhibit caspase activity. Therefore, when cells lose K(+) in response to UVB, caspases are activated and cells go into apoptosis. This supports our hypothesis that K(+) inhibits caspase activity.

  17. Keratinocyte Growth Factor-2 on the Proliferation of Corneal Epithelial Stem Cells in Rabbit Alkali Burned Cornea

    Institute of Scientific and Technical Information of China (English)

    Liu; Yongping; Shuqi; Huang; Jianxian; Lin; Wenxin; Zhang

    2007-01-01

    Purpose: To determine whether the topical application of keratinocyte growth factor-2 (KGF-2) can enhance corneal epithelial healing in rabbit alkali burned cornea. In addition, the distribution and proliferation of corneal epithelial stem cells in KGF-2-treated and control corneas were investigated to explain their mechanisms of effects on the epithelium.Methods: Twenty-four New Zealand eyes were divided into four groups, treated with KGF-2 solution (1, 50, 100 μg/ml) and PBS solution. Eighth millimeter filter paper discs, produced by standard paper punch, were soaked for 15 sec in 0.5N NaOH solution. The alkali-soaked discs were applied to the central cornea, centered on the pupil and held gently in position with forceps for 1 min. The cornea was finally irrigated over 1 min with 100 ml balanced salt solution (BSS). Keratinocyte growth factor-2 was then applied topically three times a day. The phosphate-buffered saline (PBS) group was served as a control. Each corneal epithelial defect was subsequently photographed every 24 hours with a slit lamp and was measured by computer-assisted digitizer. In each group, two rabbits were sacrificed for light microscopic examination after the interval of 7, 14 and 21 days. Meanwhile, the cornea epithelium was examined by immunohistochemistry for P63, AE5, EGFR.Results: Topical application of 10 μg/ml to 100 μg/ml KGF-2 significantly accelerated corneal epithelial wound healing when compared with controls. After 24 hours,epithelial healing rate of the 100 μg/ml KGF-2 group and the PBS treated group was (74±6)% and (40±8)% (P < 0.05). After 48 hours, the rate of the C group was (94±6)%, whereas in the control group it was (73±12)% (P < 0.05). Epithelial defects were often recurrent, which happened only two times in the 100 μg/ml KGF-2-treated group, but many times in the control group. In the corneal epithelial stem cell analysis, the number of the P63 positive cells was higher in the KGF-2-treated corneal

  18. [The causes of necrobiosis and apoptosis of corneal epithelial cells during primary acquired keratoconus].

    Science.gov (United States)

    Ziangirova, G G; Antonova, O V

    2002-01-01

    We studied 56 biopsy samples of conjunctiva and 50 corneal discs excised from 28 patients with acquired keratoconus cornea. The conjunctivas in all biopsy samples showed various stages of immune inflammation. Necrobiotic changes have been revealed in epithelium of the corneal discs going by the pathways of apoptosis--programmed cell death--and oncosis--initial edematic stage of necrobiosis. At the stage of acute inflammation they are due to cytotoxic effect of the lymphocytes, monocytes, and macrophages. Antibody-dependent cytotoxicity mediated by plasma and lymphoid cells predominates at this stage. At the reparative stage of inflammation ischemia, an inductor of apoptosis and oncosis, underlies necrobiotic changes in corneal epithelium.

  19. γδ T Cells Are Necessary for Platelet and Neutrophil Accumulation in Limbal Vessels and Efficient Epithelial Repair after Corneal Abrasion

    OpenAIRE

    Li, Zhijie; Burns, Alan R.; Rumbaut, Rolando E.; Smith, C. Wayne

    2007-01-01

    Corneal epithelial abrasion in C57BL/6 mice induces an inflammatory response with peak accumulation of neutrophils in the corneal stroma within 12 hours. Platelets localize in the limbal vessels throughout the same time course as neutrophils and contribute to wound healing because antibody-dependent depletion of platelets retards epithelial division and wound closure. In the present study, T cells in the limbal epithelium were found to predominantly express the γδ T-cell receptor (TCR). Corne...

  20. Stromal-epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model.

    Science.gov (United States)

    Kobayashi, Takeshi; Shiraishi, Atsushi; Hara, Yuko; Kadota, Yuko; Yang, Lujun; Inoue, Tomoyuki; Shirakata, Yuji; Ohashi, Yuichi

    2015-06-01

    Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal-epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal-epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin-eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two-three layers after five days and five-seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-β1, TGF-β2, and TGF-β3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-β family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal-epithelial cell

  1. The Protective Role of Hyaluronic Acid in Cr(VI-Induced Oxidative Damage in Corneal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Wei Wu

    2017-01-01

    Full Text Available Cr(VI exposure could produce kinds of intermediates and reactive oxygen species, both of which were related to DNA damage. Hyaluronan (HA has impressive biological functions and was reported to protect corneal epithelial cells against oxidative damage induced by ultraviolet B, benzalkonium chloride, and sodium lauryl sulfate. So the aim of our study was to investigate HA protection on human corneal epithelial (HCE cells against Cr(VI-induced toxic effects. The HCE cell lines were exposed to different concentrations of K2Cr2O7 (1.875, 3.75, 7.5, 15.0, and 30 μM or a combination of K2Cr2O7 and 0.2% HA and incubated with different times (15 min, 30 min, and 60 min. Our data showed that Cr(VI exposure could cause decreased cell viability, increased DNA damage, and ROS generation to the HCE cell lines. But incubation of HA increased HCE cell survival rates and decreased DNA damage and ROS generation induced by Cr(VI in a dose- and time-dependent manner. We report for the first time that HA can protect HCE cells against the toxicity of Cr(VI, indicating that it will be a promising therapeutic agent to corneal injuries caused by Cr(VI.

  2. Ambient fine particulate matters induce cell death and inflammatory response by influencing mitochondria function in human corneal epithelial cells.

    Science.gov (United States)

    Park, Eun-Jung; Chae, Jae-Byoung; Lyu, Jungmook; Yoon, Cheolho; Kim, Sanghwa; Yeom, Changjoo; Kim, Younghun; Chang, Jaerak

    2017-11-01

    Ambient fine particulate matter (AFP) is a main risk factor for the cornea as ultraviolet light. However, the mechanism of corneal damage following exposure to AFP has been poorly understood. In this study, we first confirmed that AFP can penetrate the cornea of mice, considering that two-dimensional cell culture systems are limited in reflecting the situation in vivo. Then, we investigated the toxic mechanism using human corneal epithelial (HCET) cells. At 24h after exposure, AFP located within the autophagosome-like vacuoles, and cell proliferation was clearly inhibited in all the tested concentration. Production of ROS and NO and secretion of pro-inflammatory cytokines were elevated in a dose-dependent manner. Additionally, conversion of LC3B from I-type to II-type and activation of caspase cascade which show autophagic- and apoptotic cell death, respectively, were observed in cells exposed to AFP. Furthermore, AFP decreased mitochondrial volume, inhibited ATP production, and altered the expression of metabolism-related genes. Taken together, we suggest that AFP induces cell death and inflammatory response by influencing mitochondrial function in HCET cells. In addition, we recommend that stringent air quality regulations are needed for eye health. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Effect of sertraline on Ca²⁺ fluxes in rabbit corneal epithelial cells.

    Science.gov (United States)

    Yeh, Jeng-Hsien; Sun, Te-Kung; Chou, Chiang-Ting; Chen, Wei-Chuan; Lee, Jenn-Kuen; Yeh, Hsiao-Chun; Liang, Wei-Zhe; Kuo, Chun-Chi; Shieh, Pochuen; Kuo, Daih-Huang; Jan, Chung-Ren

    2015-04-30

    The effect of sertraline, a selective serotonin reuptake inhibitor (SSRI), on cytosolic free Ca²⁺ concentrations ([Ca²⁺](i)) in a rabbit corneal epithelial cell line (SIRC) is unclear. This study explored whether sertraline changed basal [Ca²⁺](i) levels in suspended SIRC cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Sertraline at concentrations between 10-100 μM increased [Ca²⁺](i) in a concentration-dependent manner. The Ca²⁺ signal was reduced by 23% by removing extracellular Ca²⁺. Sertraline induced Mn²⁺ influx, leading to quench of fura-2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A₂ inhibitor aristolochic acid, but not by store-operated Ca²⁺ channel blockers and protein kinase C/A modulators. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin, cyclopiazonic acid or 2,5-di-tert-butylhydroquinone greatly inhibited sertraline-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 abolished sertraline-induced [Ca²⁺](i) rise. At concentrations of 5-50 μM, sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of 25 μM sertraline was not reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in SIRC cells, sertraline induced [Ca²⁺](i) rises by causing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A₂-sensitive Ca²⁺ channels. Sertraline-caused cytotoxicity was mediated by Ca²⁺-independent pathways.

  4. The anti-inflammatory effects of asiatic acid in lipopolysaccharide-stimulated human corneal epithelial cells

    Science.gov (United States)

    Chen, Hao; Hua, Xiao-Min; Ze, Bai-Chen; Wang, Bin; Wei, Li

    2017-01-01

    AIM To investigate the anti-inflammatory effects of asiatic acid (AA) on lipopolysaccharide (LPS)-induced inflammatory response in human corneal epithelial cells (HCECs). METHODS Cell viability was measured using a cell counting kit-8 (CCK-8) assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the mRNA expression of interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-β (TGF-β) in HCECs. Intracellular reactive oxygen species (ROS) was measured using the ROS assay kit. Glutathione (GSH) concentration was measured using the total GSH assay kit. Akt1 and Akt phosphorylation (p-Akt1) levels were measured by Western blotting and immunofluorescence. RESULTS AA induced toxicity at high concentrations and significantly stimulated the proliferation of HCECs at concentrations of 20 µmol/L for 1h. LPS at concentrations of 300 ng/mL for 1h significantly stimulated the mRNA expression of IL-8, IL-6, IL-1β, TNF-α, and TGF-β in HCECs, while the stimulation effects were significantly inhibited by AA (20 µmol/L). In addition, AA was found to decrease the content of ROS, increase GSH generation, and also inhibit LPS-induced p-Akt in HCECs. CONCLUSION AA decreases the generation of inflammatory factors IL-8, IL-6, IL-1β, TNF-α, and TGF-β in LPS-stimulated HCECs. AA significantly inhibites the intracellular concentrations of ROS and increases GSH generation. AA also inhibites LPS-induced p-Akt in HCECs. These findings reveal that AA has anti-inflammation effects in LPS-stimulated HCECs.

  5. Research progress in the metabolism of corneal epithelial cells%角膜上皮细胞代谢的研究进展

    Institute of Scientific and Technical Information of China (English)

    马佰凯; 何昕; 李炜

    2014-01-01

    角膜上皮层位于角膜表面,外邻泪膜,内与角膜前弹力层相连。角膜上皮细胞代谢所需营养及氧分主要通过泪膜、房水和角膜缘毛细血管运送。正常的角膜上皮细胞代谢是维持角膜上皮细胞正常增殖与分化状态的关键。角膜上皮细胞代谢异常可导致上皮损伤或变性,是多种角膜疾病的病理基础。本文就近年来关于角膜上皮细胞代谢相关的组织结构、营养来源、细胞增殖分化以及相关疾病的研究进展进行综述。%Corneal epithelium is located in the surface layer of the cornea, with tear film outside and Bowman's layer inside. The nutrient and oxygen for the metabolism of corneal epithelial cells is transmitted by tear film, anterior aqueous humor and corneal limbal capillary. Metabolism of the corneal epithelial cells is tightly correlated with its proliferation and differentiation. The abnormal metabolism of corneal epithelial cells can cause epithelial damage or dystrophy, which is the pathological basis of many corneal diseases. This review summarizes recent research progress regarding corneal epithelial cell metabolism related tissue structure, origin of nutrient, cell proliferation and differentiation and diseases related to corneal epithelial cell metabolism.

  6. Selenium-binding lactoferrin is taken into corneal epithelial cells by a receptor and prevents corneal damage in dry eye model animals.

    Science.gov (United States)

    Higuchi, Akihiro; Inoue, Hiroyoshi; Kaneko, Yoshio; Oonishi, Erina; Tsubota, Kazuo

    2016-11-11

    The ocular surface is strongly affected by oxidative stress, which causes many ocular diseases including dry eye. Previously, we showed that selenium compounds, e.g., selenoprotein P and Se-lactoferrin, were candidates for treatment of dry eye. This paper shows the efficacy of Se-lactoferrin for the treatment of dry eye compared with Diquas as a control drug using two dry eye models and incorporation of lactoferrin into corneal epithelial cells via lactoferrin receptors. We show the efficacy of Se-lactoferrin eye drops in the tobacco smoke exposure rat dry eye model and short-term rabbit dry eye model, although Diquas eye drops were only effective in the short-term rabbit dry eye model. These results indicate that Se-lactoferrin was useful in the oxidative stress-causing dry eye model. Se-lactoferrin was taken into corneal epithelium cells via lactoferrin receptors. We identified LRP1 as the lactoferrin receptor in the corneal epithelium involved in lactoferrin uptake. Se-lactoferrin eye drops did not irritate the ocular surface of rabbits. Se-lactoferrin was an excellent candidate for treatment of dry eye, reducing oxidative stress by a novel mechanism.

  7. The effect of environmental factors on the response of human corneal epithelial cells to nanoscale substrate topography.

    Science.gov (United States)

    Teixeira, Ana I; McKie, George A; Foley, John D; Bertics, Paul J; Nealey, Paul F; Murphy, Christopher J

    2006-07-01

    We have previously shown that human corneal epithelial cells sense and react to nanoscale substrate topographic stimuli [Teixeira AI, Abrams GA, Bertics PJ, Murphy CJ, Nealey PF. Epithelial contact guidance on well-defined micro- and nanostructured substrates. J Cell Sci 2003;116(10):1881-92; Karuri NW, Liliensiek S, Teixeira AI, Abrams G, Campbell S, Nealey PF, et al. Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells. J Cell Sci 2004;117(15):3153-64]. Here we demonstrate that cellular responses to nanoscale substrate topographies are modulated by the context in which these stimuli are presented to cells. In Epilife medium, cells aligned preferentially in the direction perpendicular to nanoscale grooves and ridges. This is in contrast to a previous study where cells cultured in DMEM/F12 medium aligned in the direction parallel to nanoscale topographic features [Teixeira AI, Abrams GA, Bertics PJ, Murphy CJ, Nealey PF. Epithelial contact guidance on well-defined micro- and nanostructured substrates. J Cell Sci 2003;116(10):1881-92]. Additionally, cell alignment in Epilife medium was dependent on pattern pitch. Cells switched from perpendicular to parallel alignment when the pitch was increased from 400 to 4,000 nm. There was a transition region (between 800 and 1,600 nm pitch) where both parallel and perpendicular alignments were favored compared to all other cellular orientations. Cells formed focal adhesions parallel to the substrate topographies in this transition region. On the nano- and microscale patterns, 400 and 4,000 nm pitch, focal adhesions were almost exclusively oriented obliquely to the topographic patterns.

  8. Dectin-1 agonist curdlan modulates innate immunity to Aspergillus fumigatus in human corneal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Cheng-Cheng; Zhu; Gui-Qiu; Zhao; Jing; Lin; Li-Ting; Hu; Qiang; Xu; Xu-Dong; Peng; Xue; Wang; Sheng; Qiu

    2015-01-01

    · AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus(A. fumigatus) in cultured human corneal epithelial cells(HCECs), and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan.·METHODS: The HCECs were stimulated by curdlan in different concentrations(50, 100, 200, 400 μg/m L) for various time. Then HCECs pretreated with or without laminarin(Dectin-1 blocker, 0.3 mg/m L) and curdlan were stimulated by A. fumigatus hyphae. The m RNA and protein production of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6) were determined by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot.· RESULTS: Curdlan stimulated m RNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at m RNA and protein levels compared with A. fumigatus hyphae stimulation group(P <0.05).Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphae stimulation. The Dectin-1blocker laminarin suppressed the m RNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae(P <0.05).· CONCLUSION: These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs.Dectin-1 is essential for the immunomodulatory effectsof curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.

  9. Dectin-1 agonist curdlan modulates innate immunity to Aspergillus fumigatus in human corneal epithelial cells

    Directory of Open Access Journals (Sweden)

    Cheng-Cheng Zhu

    2015-08-01

    Full Text Available AIM: To explore the immunomodulatory effects of curdlan on innate immune responses against Aspergillus fumigatus (A. fumigatus in cultured human corneal epithelial cells (HCECs, and whether C-type lectin receptor Dectin-1 mediates the immunomodulatory effects of curdlan.METHODS:The HCECs were stimulated by curdlan in different concentrations (50, 100, 200, 400 μg/mL for various time. Then HCECs pretreated with or without laminarin (Dectin-1 blocker, 0.3 mg/mL and curdlan were stimulated by A. fumigatus hyphae. The mRNA and protein production of tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6 were determined by real-timequantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The protein level of Dectin-1 was measured by Western blot.RESULTS: Curdlan stimulated mRNA expression of TNF-α and IL-6 in a dose and time dependent manner in HCECs. Curdlan pretreatment before A. fumigatus hyphae stimulation significantly enhanced the expression of TNF-α and IL-6 at mRNA and protein levels compared with A. fumigatus hyphae stimulation group (P<0.05. Both curdlan and A. fumigatus hyphae up-regulated Dectin-1 protein expression in HCECs, and Dectin-1 expression was elevated to 1.5- to 2-fold by curdlan pretreatment followed hyphaestimulation. The Dectin-1 blocker laminarin suppressed the mRNA expression and protein production of TNF-α and IL-6 induced by curdlan and hyphae (P<0.05.CONCLUSION:These findings demonstrated that curdlan pretreatment enhanced the inflammatory response induced by A. fumigatus hyphae in HCECs. Dectin-1 is essential for the immunomodulatory effects of curdlan. Curdlan may have high clinical application values in fungal keratitis treatment.

  10. Plasma polymer-coated contact lenses for the culture and transfer of corneal epithelial cells in the treatment of limbal stem cell deficiency.

    Science.gov (United States)

    Brown, Karl David; Low, Suet; Mariappan, Indumathi; Abberton, Keren Maree; Short, Robert; Zhang, Hong; Maddileti, Savitri; Sangwan, Virender; Steele, David; Daniell, Mark

    2014-02-01

    Extensive damage to the limbal region of the cornea leads to a severe form of corneal blindness termed as limbal stem cell deficiency (LSCD). Whereas most cases of corneal opacity can be treated with full thickness corneal transplants, LSCD requires stem cell transplantation for successful ocular surface reconstruction. Current treatments for LSCD using limbal stem cell transplantation involve the use of murine NIH 3T3 cells and human amniotic membranes as culture substrates, which pose the threat of transmission of animal-derived pathogens and donor tissue-derived cryptic infections. In this study, we aimed to produce surface modified therapeutic contact lenses for the culture and delivery of corneal epithelial cells for the treatment of LSCD. This approach avoids the possibility of suture-related complications and is completely synthetic. We used plasma polymerization to deposit acid functional groups onto the lenses at various concentrations. Each surface was tested for its suitability to promote corneal epithelial cell adhesion, proliferation, retention of stem cells, and differentiation and found that acid-based chemistries promoted better cell adhesion and proliferation. We also found that the lenses coated with a higher percentage of acid functional groups resulted in a higher number of cells transferred onto the corneal wound bed in rabbit models of LSCD. Immunohistochemistry of the recipient cornea confirmed the presence of autologous, transplanted 5-bromo-2'-deoxyuridine (BrdU)-labeled cells. Hematoxylin staining has also revealed the presence of a stratified epithelium at 26 days post-transplantation. This study provides the first evidence for in vivo transfer and survival of cells transplanted from a contact lens to the wounded corneal surface. It also proposes the possibility of using plasma polymer-coated contact lenses with high acid functional groups as substrates for the culture and transfer of limbal cells in the treatment of LSCD.

  11. Trehalose-Based Eye Drops Preserve Viability and Functionality of Cultured Human Corneal Epithelial Cells during Desiccation

    Directory of Open Access Journals (Sweden)

    Aneta Hill-Bator

    2014-01-01

    Full Text Available This paper presents the evaluation of cytoprotective ability of trehalose-based eye drops in comparison with commercially available preparations during the experimental desiccation of cultured human corneal epithelial cells. Cultured human corneal epithelial cells (hCEC underwent incubation with 7 different, commercially available medicaments used commonly in dry eye syndrome treatment, followed by desiccation trial performed on air under the flow hood for 5, 15, 30, and 45 minutes. Cell viability was quantified by live/dead fluorescent assay, while the presence of apoptotic cells was estimated by immunofluorescent staining for active caspase 3 protein. The preservation of membrane functions was evaluated using neutral red staining, while the preservation of proper morphology and phenotype was determined by fluorescent staining for actin filaments, nuclei, and p63 protein. The trehalose-based eye drops showed the highest efficiency in prevention of cell death from desiccation; moreover, this preparation preserved the normal cellular morphology, functions of cell membrane, and proliferative activity more effectively than other tested medicaments.

  12. [Research on effects of vitamin A palmitate on repair of mechanical corneal epithelial defects and conjunctival goblet cells in rabbits].

    Science.gov (United States)

    Qiu, Xiao-di; Gong, Lan; Chen, Min-jie

    2010-02-01

    Randomized controlled experimental study to investigate the influence of vitamin A palmitate and bovine recombinant basic fibroblast growth factor (bFGF) on repair of mechanical corneal epithelial defects, conjunctival epithelial cells and goblet cells in rabbits. One hundred and twenty New Zealand rabbits (all males) were selected to establish the mechanical corneal epithelial defects models (scratching out a round area with the diameter of 8 mm in the centre of cornea). Forty eight New Zealand rabbits were randomly divided into 4 groups: group A used lincomycin hydrochloride eye drops (LED) after the model had been established; group B used vitamin A palmitate eye gel and LED; group C used recombinant bFGF eye gel and LED; group D used vitamin A palmitate eye gel, bFGF eye gel and LED. Photo slit lamp examination and measurement of repaired area were performed on day 0, day 1, day 4 and day 7; transmission electron microscopy, histological microscope examination and impression cytology were performed on day 0, day 1, day 4 and day 7 to analysis the morphology and repairment of corneal epithelium, conjunctival epithelial cells and the goblet cells. The variants were tested using analysis of variance and Tukey's test. Statistic analysis showed that on day 1, the size of areas of repaired corneal epithelium was: group A(53.512 +/- 18.850) mm(2), group B (92.194 +/- 14.367) mm(2), group C (89.779 +/- 20.535) mm(2), group D (127.816 +/- 16.379) mm(2). The difference in size of repaired areas between different groups was statistically significant (F = 17.663, P = 0.000), with exception of the difference between groups B and C (P = 0.995). Conjunctival impression cytology showed that, the average number of conjunctival goblet cells per 740 microm x 550 microm at day 1 was decreased, group A (10.083 +/- 4.441), group B (10.667 +/- 3.551), group C (9.583 +/- 4.502), group D (9.167 +/- 5.606). The difference between these four groups was not significant (F = 0.239, P = 0

  13. Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence

    Directory of Open Access Journals (Sweden)

    Natalie J. Dorà

    2015-11-01

    Full Text Available The limbal epithelial stem cell (LESC hypothesis proposes that LESCs in the corneal limbus maintain the corneal epithelium both during normal homeostasis and wound repair. The alternative corneal epithelial stem cell (CESC hypothesis proposes that LESCs are only involved in wound repair and CESCs in the corneal epithelium itself maintain the corneal epithelium during normal homeostasis. We used tamoxifen-inducible, CreER-loxP lineage tracing to distinguish between these hypotheses. Clones of labelled cells were induced in adult CAGG-CreER;R26R-LacZ reporter mice and their distributions analysed after different chase periods. Short-lived clones, derived from labelled transient amplifying cells, were shed during the chase period and long-lived clones, derived from stem cells, expanded. At 6 weeks, labelled clones appeared at the periphery, extended centripetally as radial stripes and a few reached the centre by 14 weeks. Stripe numbers depended on the age of tamoxifen treatment. Stripes varied in length, some were discontinuous, few reached the centre and almost half had one end at the limbus. Similar stripes extended across the cornea in CAGG-CreER;R26R-mT/mG reporter mice. The distributions of labelled clones are inconsistent with the CESC hypothesis and support the LESC hypothesis if LESCs cycle between phases of activity and quiescence, each lasting several weeks.

  14. Apoptosis of Corneal Epithelial Cells Caused by Ultraviolet B-induced Loss of K(+) is Inhibited by Ba(2.).

    Science.gov (United States)

    Glupker, Courtney D; Boersma, Peter M; Schotanus, Mark P; Haarsma, Loren D; Ubels, John L

    2016-07-01

    UVB exposure at ambient outdoor levels triggers rapid K(+) loss and apoptosis in human corneal limbal epithelial (HCLE) cells cultured in medium containing 5.5 mM K(+), but considerably less apoptosis occurs when the medium contains the high K(+) concentration that is present in tears (25 mM). Since Ba(2+) blocks several K(+) channels, we tested whether Ba(2+)-sensitive K(+) channels are responsible for some or all of the UVB-activated K(+) loss and subsequent activation of the caspase cascade and apoptosis. Corneal epithelial cells in culture were exposed to UVB at 80 or 150 mJ/cm(2). Patch-clamp recording was used to measure UVB-induced K(+) currents. Caspase-activity and TUNEL assays were performed on HCLE cells exposed to UVB followed by incubation in the presence or absence of Ba(2+). K(+) currents were activated in HCLE cells following UVB-exposure. These currents were reversibly blocked by 5 mM Ba(2+). When HCLE cells were incubated with 5 mM Ba(2+) after exposure to UVB, activation of caspases-9, -8, and -3 and DNA fragmentation were significantly decreased. The data confirm that UVB-induced K(+) current activation and loss of intracellular K(+) leads to activation of the caspase cascade and apoptosis. Extracellular Ba(2+) inhibits UVB-induced apoptosis by preventing loss of intracellular K(+) when K(+) channels are activated. Ba(2+) therefore has effects similar to elevated extracellular K(+) in protecting HCLE cells from UVB-induced apoptosis. This supports our overall hypothesis that elevated K(+) in tears contributes to protection of the corneal epithelium from adverse effects of ambient outdoor UVB.

  15. Evaluation of Biomarkers of Inflammation in Response to Benzalkonium Chloride on Corneal and Conjunctival Epithelial Cells

    Science.gov (United States)

    Chen, Dongmei; Asbell, Penny A.

    2009-01-01

    Abstract Purpose Most eye drops contain preservatives; benzalkonium chloride (BAK) is most common. Recent data demonstrated BAK adding to toxicity. BAK is degraded into hydrogen peroxide (H2O2), which in even small amounts is known to be an irritant. Increased toxicity should cause localized inflammation with increased elaboration of inflammatory biomarkers. To evaluate the inflammation BAK causes to the ocular surface, enzyme linked immunosorbant assays (ELISAs) were utilized to quantify the levels of inflammatory biomarkers in response to BAK and/or H2O2. Methods Immortalized human conjunctival and corneal epithelial cells were exposed to: BAK (0.001%–0.1%), hydrogen peroxide (H2O2) (0.01%–0.1%), and cell media for 1 h. Cytokine quantification was performed via enzyme-linked immunosorbent assays [ELISAs]). Additional experimentation was performed in which testing solutions were replaced with media after 1 h and the resulting supernatants quantified after 24 h. Results BAK induced significant amounts of interleukin (IL-) 1 and tumor necrosis factor (TNF), but only moderate amounts of C-reactive protein (CRP), IL- 10 and 12, and H2O2. Lower concentrations of BAK induced proportionally less elaboration. Replacing the test solutions with media and providing 23 h for cytokine elaboration significantly increased TNF, but not IL-1. Lipopolysaccharide (LPS) positive controls induced substantial elaboration/release of both IL-1 and TNF as did in increasing the exposure to the full 24 h. Conclusions After 1 h of exposure, BAK increased quantities of all biomarkers. The biomarkers in decreasing order of induction/upregulation were: TNF ≥ IL-1 ≥ IL-12 ≥ IL-10 ≥ CRP. Even low concentrations caused some degree of inflammation. Replacing the testing solution with media and providing 23 h for cytokine elaboration, significantly increased the elaboration/release of TNF, but not IL-1, as compared to the 1-h BAK exposure. Whereas increasing the exposure to the full 24 h

  16. Indoleamine 2,3-Dioxygenase Is Involved in the Inflammation Response of Corneal Epithelial Cells to Aspergillus fumigatus Infections.

    Directory of Open Access Journals (Sweden)

    Nan Jiang

    Full Text Available Indoleamine 2,3-dioxygenase (IDO, which is mainly expressed in activated dendritic cells, is known as a regulator of immune responses. However, the role of IDO in immune responses against fungal corneal infection has not been investigated. To evaluate the regulatory mechanisms of IDO in fungal inflammation, we resorted to human corneal epithelial cells (HCECs, known as the first barrier of cornea against pathogenic microorganisms. We found that IDO was significantly up-regulated in corneal epithelium infected with Aspergillus fumigatus (A. fumigatus and HCECs incubated with spores of A. fumigatus. Furthermore, IDO inhibitor (1-methyltryptophan, 1-MT enhanced inflammatory cytokines IL-1β and IL-6 expression which were up-regulated by A. fumigatus spores infection. Dectin-1, as one of the important C-type lectin receptors, can identify β-glucan, and mediate fungal innate immune responses. In the present study, pre-treatment with curdlan, a Dectin-1 agonist, further enhanced IDO expression compared with A. fumigatus stimulation. While laminarin, the Dectin-1 specific inhibitor, partially inhibited IDO expression stimulated by A. fumigatus. Further studies demonstrated inhibition of IDO activity amplified the expressions of inflammatory cytokines IL-1β and IL-6 induced by activation of Dectin-1. These results suggested that IDO was involved in the immune responses of fungal keratitis. The activation of Dectin-1 may contribute to A. fumigatus spores-induced up-regulation of IDO.

  17. Glycosaminoglycans are involved in pathogen adherence to corneal epithelial cells differently for Gram-positive and Gram-negative bacteria

    Directory of Open Access Journals (Sweden)

    Beatriz García

    2016-11-01

    Full Text Available The epithelium of the cornea is continuously exposed to pathogens, and adhesion to epithelial cells is regarded as an essential first step in bacterial pathogenesis. In this article, the involvement of glycosaminoglycans in the adhesion of various pathogenic bacteria to corneal epithelial cells is analyzed. All microorganisms use glycosaminoglycans as receptors, but arranged in different patterns depending on the Gram-type of the bacterium. The heparan sulfate chains of syndecans are the main receptors, though other molecular species also seem to be involved, particularly in Gram-negative bacteria. Adherence is inhibited differentially by peptides, including heparin binding sequences, indicating the participation of various groups of Gram-positive and -negative adhesins. The length of the saccharides produces a major effect, and low molecular weight chains inhibit the binding of Gram-negative microorganisms but increase the adherence of Gram-positives. Pathogen adhesion appears to occur preferentially through sulfated domains, and is very dependent on N- and 6-O-sulfation of the glucosamine residue and, to a lesser extent, 2-O sulfation of uronic acid. These data show the differential use of corneal receptors, which could facilitate the development of new anti-infective strategies.

  18. The Effects of Triptolide on HLA Antigens Expression of Corneal Epithelial Cells Induced by Interferon-γin Vitro

    Institute of Scientific and Technical Information of China (English)

    Qi Zhao; Yiezi Liu; Quanfu Li

    2000-01-01

    Objective: To observe the effects of immunosuppressants triptolide (TL) and cyclosporine A (CSA) on HLA antigens expression induced by interferon-γ(INF -γ) in vitro.Method: By using an indirect immunofluorescent method and analysing with ACAS-570, the abnormal HLA antigen expression of cultured corneal epithelial cells was induced by INF-γ. After incubation with one of the immunosuppressants (CSA, TL) for 72 hrs, the amount of HLA-A BC and HLA-DR antigens was measured.Result: There was no significant difference ( P > 0.05) between the group with CSA and the positive control group without CSA. In contrast to CSA, TL dramatically inhibited INF-γ induced expression of HLA antigens of corneal epithelial cells (P<0.001), compared with the control group without TL.Conclusion: TL had direct inhibition on the expression of HLA-ABCand HLA-DR antigens induced by INF-γin vitro, while CSA had no obvious inhibition. Eye Science 2000; 16:34 ~ 37.

  19. Effect of polysaccharide of dendrobium candidum on proliferation and apoptosis of human corneal epithelial cells in high glucose.

    Science.gov (United States)

    Li, Qiangxiang; Chen, Jing; Li, Yajia; Chen, Ting; Zou, Jing; Wang, Hua

    2017-08-01

    The aim of the study was to observe the effect of polysaccharide of dendrobium candidum (PDC) and high glucose on proliferation, apoptosis of human corneal epithelial cells (HCEC). The MTT method was used to screen and take the optimal high-glucose concentration, treatment time, and PDC concentration using HCEC and divide it into 4 groups: control group (C), high glucose group (HG), PDC group, and HG + PDC group. We observed and compared the effect of the 4 groups on HCEC proliferation by MTT, apoptosis by Annexin V-FITC/PI double fluorescent staining and flow cytometry (FCM), and expression of bax mRNA and bcl-2 mRNA by RT-qPCR. Compared with the control group, proliferative activity of HCEC cells was reduced; the cells apoptosis ratio was increased; the expression of bax mRNA was increased, and the expression of bcl-2 mRNA was reduced in the HG group. Proliferative activity of HCEC cells in the PDC group was increased, and the expression of bcl-2 mRNA was increased but that of bax mRNA was decreased. Proliferative activity of HCEC cells in the HG + PDC group was increased, but it could not restore to the normal level; the expression of bax mRNA was significantly decreased but the expression of bcl-2 mRNA was significantly increased. Our results demonstrate that high glucose can inhibit proliferative activity and induce apoptosis of HCEC. PDC can improve the proliferative activity of HCEC cells under the high glucose environment and reduce the apoptosis of cells by regulating the expression of bax and bcl-2. PDC play a very important role on protecting and repairing of corneal epithelial cells damage in high glucose.

  20. Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Hong [Department of Ophthalmology, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012 (China); The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012 (China); Wu, Xinyi, E-mail: xywu8868@163.com [Department of Ophthalmology, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012 (China)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-{beta}. Black-Right-Pointing-Pointer Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. Black-Right-Pointing-Pointer Hypoxia inhibits Acanthamoeba-induced the activation of NF-{kappa}B and ERK1/2 in HCECs. Black-Right-Pointing-Pointer Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. Black-Right-Pointing-Pointer LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-{beta} (IFN-{beta}) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-{beta}. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-{kappa}B and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88

  1. Evaluation of a human corneal epithelial cell line as an in vitro model for assessing ocular irritation.

    Science.gov (United States)

    Kruszewski, F H; Walker, T L; DiPasquale, L C

    1997-04-01

    A human corneal epithelial cell line, 10.014 pRSV-T (HCR-T cells), has been used to develop a three-dimensional in vitro model of the human corneal epithelium (HCE-T model). HCE-T cells form a stratified culture when grown at the air-liquid interface on a collagen membrane in serum-free medium. This model served as the basis for assays which supported the ocular irritancy assessment of water-soluble test substances. Cellular alterations in the HCE-T model were measured following 5-min topical exposures to 20 chemicals [listed in the European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) Reference Chemicals Data Bank] and 25 surfactant-based product formulations [utilized in the Cosmetic, Toiletry, and Fragrance Association (CTFA) Alternatives Program Phase III]. In vitro assays used were transepithelial permeability to sodium fluorescein (TEP) and transepithelial electrical resistance (TER). These measured alterations in the barrier function of this corneal epithelial equivalent. Barrier function is a well-developed property in the HCE-T model that supports the mechanistic relevance of these assays. In vitro data, averaged from replicate assays, were compared to respective Draize rabbit eye irritation data from the publicly available ECETOC and CTFA databases using linear regression with Pearson's correlation analysis. For chemicals, Pearson's correlation coefficients, r, from comparisons of Draize maximum average scores (MAS) to TEP and TER data were 0.71 and 0.55, respectively. For product formulations, Pearson's correlation coefficients from comparisons of Draize MAS to TEP and TER data were 0.86 and 0.80, respectively. Data indicated that barrier function alterations in the HCE-T model correlated with ocular irritancy and corneal toxicity. While the irritancy of the chemicals tested was effectively assessed only by the TEP assay, that for the surfactant-based product formulations was effectively assessed by both the TEP and TER assays. Results

  2. Brm inhibits the proliferative response of keratinocytes and corneal epithelial cells to ultraviolet radiation-induced damage.

    Directory of Open Access Journals (Sweden)

    Nur Mohammad Monsur Hassan

    Full Text Available Ultraviolet radiation (UV from sunlight is the primary cause of skin and ocular neoplasia. Brahma (BRM is part of the SWI/SNF chromatin remodeling complex. It provides energy for rearrangement of chromatin structure. Previously we have found that human skin tumours have a hotspot mutation in BRM and that protein levels are substantially reduced. Brm-/- mice have enhanced susceptibility to photocarcinogenesis. In these experiments, Brm-/- mice, with both or a single Trp53 allele were exposed to UV for 2 or 25 weeks. In wild type mice the central cornea and stroma became atrophic with increasing time of exposure while the peripheral regions became hyperplastic, presumably as a reparative process. Brm-/-, Trp53+/-, and particularly the Brm-/- Trp53+/- mice had an exaggerated hyperplastic regeneration response in the corneal epithelium and stroma so that the central epithelial atrophy or stromal loss was reduced. UV induced hyperplasia of the epidermis and corneal epithelium, with an increase in the number of dividing cells as determined by Ki-67 expression. This response was considerably greater in both the Brm-/- Trp53+/+ and Brm-/- Trp53+/- mice indicating that Brm protects from UV-induced enhancement of cell division, even with loss of one Trp53 allele. Cell division was disorganized in Brm-/- mice. Rather than being restricted to the basement membrane region, dividing cells were also present in the suprabasal regions of both tissues. Brm appears to be a tumour suppressor gene that protects from skin and ocular photocarcinogenesis. These studies indicate that Brm protects from UV-induced hyperplastic growth in both cutaneous and corneal keratinocytes, which may contribute to the ability of Brm to protect from photocarcinogenesis.

  3. Dependence of Regulatory Volume Decrease on Transient Receptor Potential Vanilloid 4 (TRPV4) Expression in Human Corneal Epithelial Cells

    Science.gov (United States)

    Pan, Zan; Yang, Hua; Mergler, Stefan; Liu, Hongshan; Tachado, Souvenir D.; Zhang, Fan; Kao, Winston W. Y.; Koziel, Henry; Pleyer, Uwe; Reinach, Peter S.

    2008-01-01

    TRPV4 is a non-selective cation channel with moderate calcium permeability, which is activated by exposure to hypotonicity. Such a stress induces regulatory volume decrease (RVD) behavior in human corneal epithelial cells (HCEC). We hypothesize that TRPV4 channel mediates RVD in HCEC. Immunohistochemistry revealed centrally and superficially concentrated TRPV4 localization in the corneal tissue. Immunocytochemical and Fluorescence Activated Cell Sorter (FACS) analyses identified TRPV4 membrane surface and cytosolic expression. RT-PCR and Western blot analyses identified TRPV4 gene and protein expression in HCEC, respectively. In addition, 4α-PDD or a 50% hypotonic medium induced up to three-fold transient intracellular Ca2+ ([Ca2+]i) increases. Following TRPV4 siRNA HCEC transfection, its protein expression level declined by 64%, which abrogated these [Ca2+]i transients. Similarly, exposure to either ruthenium red or Ca2+-free Ringer's solution also eliminated this response. In these transfected cells, RVD declined by 51% whereas in the non-transfected counterpart, ruthenium red and Ca2+-free solution inhibited RVD by 54% and 64%, respectively. In contrast, capsazepine, a TRPV1 antagonist, failed to suppress [Ca2+]i transients and RVD. TRPV4 activation contributes to RVD since declines in TRPV4 expression and activity are associated with suppression of this response. In conclusion, there is TRPV4 functional expression in HCEC. PMID:18355916

  4. Comparison of cytotoxicities and wound healing effects of diquafosol tetrasodium and hyaluronic acid on human corneal epithelial cells

    Science.gov (United States)

    Lee, Jong Heon; Lee, Jong Soo; Kim, Sujin

    2017-01-01

    This study aimed to compare the cellular toxicities of three clinically used dry eye treatments; 3% diquafosol tetrasodium and hyaluronic acid at 0.3 and 0.18%. A methyl thiazolyltetrazoiun (MTT)-based calorimetric assay was used to assess cellular proliferation and a lactate dehydrogenase (LDH) leakage assay to assess cytotoxicity, using Human corneal epithelial cells (HCECs) exposed to 3% diquafosol tetrasodium, 0.3% hyaluronic acid (HA), or 0.18% HA or 1, 6 or 24 h. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy, and wound widths were measured 24 h after confluent HCECs were scratched. Diquafosol had a significant, time-dependent, inhibitory effect on HCEC proliferation and cytotoxicity. HCECs treated with diquafosol detached more from the bottoms of dishes and damaged cells showed degenerative changes, such as, reduced numbers of microvilli, vacuole formation, and chromatin of the nuclear remnant condensed along the nuclear periphery. All significantly stimulated reepithelialization of HCECs scratched, which were less observed in diquafosol. Therefore, epithelial toxicity should be considered after long-term usage of diquafosol and in overdose cases, especially in dry eye patients with pre-existing punctated epithelial erosion. PMID:28280412

  5. [Macroscopic observations on corneal epithelial wound healing in the rabbit].

    Science.gov (United States)

    Hayashi, K

    1991-02-01

    A newly-developed macroscope was applied to observe the healing process of corneal epithelial wound in vivo. After removing epithelium of the central cornea, the changes of the corneal surface were observed with the macroscope and the findings were compared with histological examinations. At 12 hours after abrasion, areas unstained with Richardson's staining (R staining) appeared. In the histological section, a single layer of regenerating epithelial cells covered the same area. At 24 and 36 hours after abrasion, the epithelial defects became smaller but surrounding epithelium was rough and showed dot-like staining with R solution. By 2 days, the epithelial defects disappeared. On macroscopic observation, the central corneal surface showed a pavement-like appearance. Histology revealed that the regenerating epithelium still consisted of one or two layers. At 3 days, dot-like stainings were present only in the center and the corneal surface appeared considerably smooth. Histology also showed that regenerating epithelium became columnar and multilayered, thereby suggesting stratification. By 7 days, the abraded corneal surface had recovered its smooth appearance. Histologic sections also demonstrated that the epithelium had regained its normal structure. Thus, using this macroscope, findings suggesting the process of epithelial migration and proliferation could be observed.

  6. Study of corneal epithelial progenitor origin and the Yap1 requirement using keratin 12 lineage tracing transgenic mice

    Science.gov (United States)

    Kasetti, Ramesh Babu; Gaddipati, Subhash; Tian, Shifu; Xue, Lei; Kao, Winston W.-Y.; Lu, Qingxian; Li, Qiutang

    2016-01-01

    Key issues in corneal epithelium biology are the mechanism for corneal epithelium stem cells to maintain the corneal epithelial homeostasis and wound healing responses, and what are the regulatory molecular pathways involved. There are apparent discrepancies about the locations of the progenitor populations responsible for corneal epithelial self-renewal. We have developed a genetic mouse model to trace the corneal epithelial progenitor lineages during adult corneal epithelial homeostasis and wound healing response. Our data revealed that the early corneal epithelial progenitor cells expressing keratin-12 originated from limbus, and gave rise to the transit amplifying cells that migrated centripetally to differentiate into corneal epithelial cells. Our results support a model that both corneal epithelial homeostasis and wound healing are mainly maintained by the activated limbal stem cells originating form limbus, but not from the corneal basal epithelial layer. In the present study, we further demonstrated the nuclear expression of transcriptional coactivator YAP1 in the limbal and corneal basal epithelial cells and its essential role for maintaining the high proliferative potential of those corneal epithelial progenitor cells in vivo. PMID:27734924

  7. Novel nuclear localization and potential function of insulin-like growth factor-1 receptor/insulin receptor hybrid in corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Yu-Chieh Wu

    Full Text Available BACKGROUND: Type I insulin-like growth factor receptor (IGF-1R and insulin receptor (INSR are highly homologous molecules, which can heterodimerize to form an IGF-1R/INSR hybrid (Hybrid-R. The presence and biological significance of the Hybrid-R in human corneal epithelium has not yet been established. In addition, while nuclear localization of IGF-1R was recently reported in cancer cells and human corneal epithelial cells, the function and profile of nuclear IGF-1R is unknown. In this study, we characterized the nuclear localization and function of the Hybrid-R and the role of IGF-1/IGF-1R and Hybrid-R signaling in the human corneal epithelium. METHODOLOGY/PRINCIPLE FINDINGS: IGF-1-mediated signaling and cell growth were examined in a human telomerized corneal epithelial (hTCEpi cell line using co-immunoprecipitation, immunoblotting and cell proliferation assays. The presence of Hybrid-R in hTCEpi and primary cultured human corneal epithelial cells was confirmed by immunofluorescence and reciprocal immunoprecipitation of whole cell lysates. We found that IGF-1 stimulated Akt and promoted cell growth through IGF-1R activation, which was independent of the Hybrid-R. The presence of Hybrid-R, but not IGF-1R/IGF-1R, was detected in nuclear extracts. Knockdown of INSR by small interfering RNA resulted in depletion of the INSR/INSR and preferential formation of Hybrid-R. Chromatin-immunoprecipitation sequencing assay with anti-IGF-1R or anti-INSR was subsequently performed to identify potential genomic targets responsible for critical homeostatic regulatory pathways. CONCLUSION/SIGNIFICANCE: In contrast to previous reports on nuclear localized IGF-1R, this is the first report identifying the nuclear localization of Hybrid-R in an epithelial cell line. The identification of a nuclear Hybrid-R and novel genomic targets suggests that IGF-1R traffics to the nucleus as an IGF-1R/INSR heterotetrameric complex to regulate corneal epithelial homeostatic

  8. Genoprotective effect of hyaluronic acid against benzalkonium chloride-induced DNA damage in human corneal epithelial cells

    Science.gov (United States)

    Wu, Han; Zhang, Huina; Wang, Changjun; Wu, Yihua; Xie, Jiajun; Jin, Xiuming; Yang, Jun

    2011-01-01

    Purpose The aim of this study was to investigate hyaluronic acid (HA) protection on cultured human corneal epithelial cells (HCEs) against benzalkonium chloride (BAC)-induced DNA damage and intracellular reactive oxygen species (ROS) increase. Methods Cells were incubated with different concentrations of BAC with or without the presence of 0.2% HA for 30 min. DNA damage to HCEs was examined by alkaline comet assay and by immunofluorescence microscopic detection of the phosphorylated form of histone variant H2AX (γH2AX) foci. ROS production was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell apoptosis was determined with annexin V staining by flow cytometry. Results HA significantly reduced BAC-induced DNA damage as indicated by the tail length (TL) and tail moment (TM) of alkaline comet assay and by γH2AX foci formation, respectively. Moreover, HA significantly decreased BAC-induced ROS increase and cell apoptosis. However, exposure to HA alone did not produce any significant change in DNA damage, ROS generation, or cell apoptosis. Conclusions BAC could induce DNA damage and cell apoptosis in HCEs, probably through increasing oxidative stress. Furthermore, HA was an effective protective agent that had antioxidant properties and could decrease DNA damage and cell apoptosis induced by BAC. PMID:22219631

  9. Hyperosmotic Stress-induced ATF-2 Activation through Polo-like Kinase 3 in Human Corneal Epithelial Cells*

    Science.gov (United States)

    Wang, Ling; Payton, Reid; Dai, Wei; Lu, Luo

    2011-01-01

    Elevated extracellular solute concentration (hyperosmotic stress) perturbs cell function and stimulates cell responses by evoking MAPK cascades and activating AP-1 transcription complex resulting in alterations of gene expression, cell cycle arrest, and apoptosis. The results presented here demonstrate that hyperosmotic stress elicited increases in ATF-2 phosphorylation through a novel Polo-like kinase 3 (Plk3) pathway in human corneal epithelial (HCE) cells. We found in hyperosmotic stress-induced HCE cells that Plk3 transferred to the nuclear compartment and was colocalized with ATF-2 in nuclei. Kinase activity of Plk3 was significantly activated by hyperosmotic stimulation. Further downstream, active Plk3 phosphorylated ATF-2 at the Thr-71 site in vivo and in vitro. Overexpression of Plk3 and its mutants enhanced hyperosmotic stress-induced ATF-2 phosphorylation. In contrast, suppression of Plk3 by knocking down Plk3 mRNA effectively diminished the effect of hyperosmotic stress-induced ATF-2 phosphorylation. The effect of hyperosmotic stress-induced activation of Plk3 on ATF-2 transcription factor function was also examined in CRE reporter-overexpressed HCE cells. Our results for the first time reveal that hyperosmotic stress can activate the Plk3 signaling pathway that subsequently regulates the AP-1 complex by directly phosphorylating ATF-2 independent from the effects of JNK and p38 activation. PMID:21098032

  10. Hyperosmotic stress-induced ATF-2 activation through Polo-like kinase 3 in human corneal epithelial cells.

    Science.gov (United States)

    Wang, Ling; Payton, Reid; Dai, Wei; Lu, Luo

    2011-01-21

    Elevated extracellular solute concentration (hyperosmotic stress) perturbs cell function and stimulates cell responses by evoking MAPK cascades and activating AP-1 transcription complex resulting in alterations of gene expression, cell cycle arrest, and apoptosis. The results presented here demonstrate that hyperosmotic stress elicited increases in ATF-2 phosphorylation through a novel Polo-like kinase 3 (Plk3) pathway in human corneal epithelial (HCE) cells. We found in hyperosmotic stress-induced HCE cells that Plk3 transferred to the nuclear compartment and was colocalized with ATF-2 in nuclei. Kinase activity of Plk3 was significantly activated by hyperosmotic stimulation. Further downstream, active Plk3 phosphorylated ATF-2 at the Thr-71 site in vivo and in vitro. Overexpression of Plk3 and its mutants enhanced hyperosmotic stress-induced ATF-2 phosphorylation. In contrast, suppression of Plk3 by knocking down Plk3 mRNA effectively diminished the effect of hyperosmotic stress-induced ATF-2 phosphorylation. The effect of hyperosmotic stress-induced activation of Plk3 on ATF-2 transcription factor function was also examined in CRE reporter-overexpressed HCE cells. Our results for the first time reveal that hyperosmotic stress can activate the Plk3 signaling pathway that subsequently regulates the AP-1 complex by directly phosphorylating ATF-2 independent from the effects of JNK and p38 activation.

  11. Treatment with retinoic acid and lens epithelial cell-conditioned medium in vitro directed the differentiation of pluripotent stem cells towards corneal endothelial cell-like cells.

    Science.gov (United States)

    Chen, Ping; Chen, Jun-Zhao; Shao, Chun-Yi; Li, Chuan-Yin; Zhang, Yi-Dan; Lu, Wen-Juan; Fu, Yao; Gu, Ping; Fan, Xianqun

    2015-02-01

    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have extensive self-renewal capacity and the potential to differentiate into all tissue-specific cell lineages, including corneal endothelial cells (CECs). They are a promising prospect for the future of regenerative medicine. The method of derivation of CECs from ESCs and iPSCs, however, remains to be elucidated. In this study, mouse ESCs and iPSCs were induced to differentiate into CECs using CEC embryonic development events as a guide. All-trans retinoic acid (RA) treatment during the embryoid body (EB) differentiation step was used to promote neural crest (NC) cell differentiation as first step and was followed by a second induction in CEC- or lens epithelial cell (LEC)-conditioned medium (CM) to ultimately generate CEC-like cells. During the corresponding differentiation stages, NC developmental markers and CEC differentiation markers were detected at the protein level using immunocytochemistry (ICC) and at the mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). During the first stage, the data indicated that 4 days of treatment with 1 μM RA starting on day 4 of EB formation favored NC cell differentiation and that plating on gelatin-coated plates led to cell migration out of the EBs. The second-stage differentiation results showed that the CM, particularly the LEC-CM, enhanced the yield of polygonal cells with CEC-specific marker expression shown by ICC and RT-qPCR. This study demonstrates that mouse ESCs and iPSCs were induced and expressed CEC differentiation markers when subjected to a two-step inducement process, suggesting that they are a promising resource for corneal endothelium failure replacement therapy in the future.

  12. First Identification of a Triple Corneal Dystrophy Association: Keratoconus, Epithelial Basement Membrane Corneal Dystrophy and Fuchs' Endothelial Corneal Dystrophy

    Directory of Open Access Journals (Sweden)

    Cosimo Mazzotta

    2014-09-01

    Full Text Available Purpose: To report the observation of a triple corneal dystrophy association consisting of keratoconus (KC, epithelial basement membrane corneal dystrophy (EBMCD and Fuchs' endothelial corneal dystrophy (FECD. Methods: A 55-year-old male patient was referred to our cornea service for blurred vision and recurrent foreign body sensation. He reported bilateral recurrent corneal erosions with diurnal visual fluctuations. He underwent corneal biomicroscopy, Scheimpflug tomography, in vivo HRT confocal laser scanning microscopy and genetic testing for TGFBI and ZEB1 mutations using direct DNA sequencing. Results: Biomicroscopic examination revealed the presence of subepithelial central and paracentral corneal opacities. The endothelium showed a bilateral flecked appearance, and the posterior corneal curvature suggested a possible concomitant ectatic disorder. Corneal tomography confirmed the presence of a stage II KC in both eyes. In vivo confocal laser scanning microscopy revealed a concomitant bilateral EBMCD with hyperreflective deposits in basal epithelial cells, subbasal Bowman's layer microfolds and ridges with truncated subbasal nerves as pseudodendritic elements. Stromal analysis revealed honeycomb edematous areas, and the endothelium showed a strawberry surface configuration typical of FECD. The genetic analysis resulted negative for TGFBI mutations and positive for a heterozygous mutation in exon 7 of the gene ZEB1. Conclusion: This is the first case reported in the literature in which KC, EBMCD and FECD are present in the same patient and associated with ZEB1 gene mutation. The triple association was previously established by means of morphological analysis of the cornea using corneal Scheimpflug tomography and in vivo HRT II confocal laser scanning microscopy.

  13. MicroRNA-145 regulates human corneal epithelial differentiation.

    Directory of Open Access Journals (Sweden)

    Sharon Ka-Wai Lee

    Full Text Available BACKGROUND: Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC epithelia containing corneal epithelial progenitor cells (CEPCs and early transit amplifying cells, and their role in corneal epithelium. METHODOLOGY/PRINCIPAL FINDINGS: Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test. This was validated by quantitative polymerase chain reaction (qPCR. Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance. As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8 expression in both human corneal epithelial cells

  14. System for tracking transplanted limbal epithelial stem cells in the treatment of corneal stem cell deficiency (Conference Presentation)

    Science.gov (United States)

    Boadi, Joseph; Matcher, Stephen; MacNeil, Sheila; Sangwan, Virender S.

    2016-04-01

    The prevailing hypothesis for the existence and healing of the avascular corneal epithelium is that this layer of cells are continually produced by stem cells in the limbus and transported onto the cornea to mature into corneal epithelium. In the event that the cornea is damaged and the limbal stem cell population is severely reduced, this condition known as Limbal Stem Cell Deficiency and can lead to blindness. There are numerous treatments but most have high long term failure rates. Most treatment methods include the transplantation of limbal stem cells into damaged limbus with hope of repopulating the region and regenerating at healthy corneal epithelium. Optical Coherence Tomography (OCT) is well known for its high resolution in vivo images. A bespoke OCT has been built to investigate the trajectories of these limbal stem cells after transplantation to see whether if they do repopulate the damaged limbus or not. In the experimentation magneto-labelling was used to track the limbal stem cells. For the magneto-labelling a mixture of limbal stem cells and cornea epithelium are cultured with super paramagnetic iron (Fe3O4) nanoparticles (20-30nm in size) for 24hours, to allow for uptake. The cells are then transplanted onto the denuded cornea. The transplanted cell mixture with the encapsulated magnetic nanoparticles is actuated with an external magnetic field 0.08T leading to a phase modulation on the signal. A Phase sensitive Magneto-motive OCT is used to locate the transplanted cells. The location of the cells with embed SPIOs were located both in 2D and 3D.

  15. Early expression of mannose-binding lectin 2 during Aspergillus fumigatus infection in human corneal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Cheng-Ye; Che; Jing-Fen; Zhang; Ji-Eun; Lee; Jing; Lin; Li-Ting; Hu; Nan; Jiang; Qian; Wang; Qiang; Xu; Gui-Qiu; Zhao

    2015-01-01

    AIM: To evaluate the early expression of mannose-binding lectin 2(MBL2) in human corneal epithelial cells(HCECs) infected by Aspergillus fumigatus(AF).METHODS: HCECs cultured in vitro with AF antigens and sampled at 0, 0.5, 1, 2, 4, 6 and 8h. The expression of MBL2 m RNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction(RT-PCR). The expression of MBL2 protein in supernatant fluid was shown by enzyme linked immunosorbent assay(ELISA). MBL2 protein in HCECs was detected by immunocytochemistry at 0 and 24 h.RESULTS: MBL2 m RNA and protein are expressed in normal HCECs. The expression of MBL2 m RNA and protein in supernatant fluid begin to increase after being stimulated with AF antigens. The most significantly peak of MBL2 m RNA is in 2h. The protein of MBL2 in supernatant fluid decrease gradually after 0.5h. The protein in HCECs expression increase after stimulation of24 h.· CONCLUSION: MBL2 receptor expressed in normal HCECs in vitro. The stimulation by AF antigens can increase the early expression of it.

  16. Fungus induces the release of IL- 8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways

    Institute of Scientific and Technical Information of China (English)

    Xu-Dong; Peng; Gui-Qiu; Zhao; Jing; Lin; Nan; Jiang; Qiang; Xu; Cheng-Cheng; Zhu; Jian-Qiu; Qu; Lin; Cong; Hui; Li

    2015-01-01

    AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the involvement of intracellular signalling pathways. METHODS: HCECs were treated with A. fumigatus hyphae antigens with different concentrations and time.The cytoplasmic calcium of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of Ca2 +-dependent protein kinase C(PKC). The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay(ELISA) and reverse transcriptase polymerase chain reaction(RT-PCR). Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to A.fumigatus hyphae antigens. RESULTS: Cells exposed to A. fumigatus hyphae antigens showed higher level of IL-8 m RNA expression and protein production. We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggers an intracellular Ca2 +flux and results in the activation of Ca2 +-dependent PKC(α, βⅠ and βⅡ) which can be attenuated by pre-treatment of cells with laminarin,suggesting that Dectin-1 signals pathway induced cytoplasmic calcium and influence the activation of PKC in HCECs. Inhibitors of Ca2 +-dependent PKC(Ro-31-8220 and Go6976) significantly abolished hyphae-induced expression of IL-8.CONCLUSION: Our findings suggest that A. fumigatushyphae-induced IL-8 expression was regulated by the activation of Dectin-1-mediated Ca2 +-dependent PKC in HCECs.

  17. Viability of corneal epithelial cells and cleavage of corneal basement after ethanol effect%乙醇对角膜上皮瓣活性及基底膜分离定位研究

    Institute of Scientific and Technical Information of China (English)

    陈颖欣; 蓝平; 高明宏; 范忠义; 宋福林; 徐旭; 于静

    2008-01-01

    Objective To evaluate the viability of corneal epithelial cells and to determine the anatomic cleavage on the epithelial basement membrane after various exposure times to 20% ethanol during epithelial flap preparation in laser-assisted subepithelial keratectomy(LASEK)in cadaver eyes.Methods Six human cadaver eyes were exposed to 20% ethanol for 20,30 and 40 seconds(2 eyes for each group),and another one eye was used as the control.PCNA staining was performed to determine the viability of corneal epithelial cells.Immunofluorescence staining using monoclonal antibodies against collagen Ⅶ,and immunohistological staining using monoclonal antibodies against laminin were performed to detect the anatomic location of the cleavage plane on the corneal epithelial flaps created by 20 seconds exposure to 20% ethanol in cadaver eyes.Results Hematoxylin and eosin staining of epithelial flaps revealed a coherent stratified epithelium.The PCNA positive rates of the epithelial cells in the flap decreased in the 20-second group,30-seconcl group and 40-second group successively.Immunohistological staining to laminin was patchy in the lifted flap and the remaining corneal basement membrane.Immunofluorescence to collagen Ⅶ,the main component of anchoring fibrils remained exclusively in the corneal bed.Conclusions Viability of the epithelial flap decreased with longer time exposure to ethanol.The cleavage plane of the ethanol-treated corneal epithelial flap is located between the lamina lucida and the lamina densa of the basement membrane where laminin forms hemidesmesome.%目的 探讨不同乙醇作用时间对角膜上皮瓣活性的影响,并确定角膜上皮瓣的解剖分离层面.方法 按标准准分子激光角膜上皮瓣下磨镶术(LASEK)方法制备7只尸体眼角膜上皮瓣,其中对照组1只眼,其余6只分为A、B、C 3个组,每组2只眼,乙醇浸润时间分别为20、30和40 S.对7只眼进行HE、增殖细胞核抗原(PCNA)、层黏连蛋白免疫组化

  18. The corneal fibrosis response to epithelial-stromal injury.

    Science.gov (United States)

    Torricelli, Andre A M; Santhanam, Abirami; Wu, Jiahui; Singh, Vivek; Wilson, Steven E

    2016-01-01

    The corneal wound healing response, including the development of stromal opacity in some eyes, is a process that often leads to scarring that occurs after injury, surgery or infection to the cornea. Immediately after epithelial and stromal injury, a complex sequence of processes contributes to wound repair and regeneration of normal corneal structure and function. In some corneas, however, often depending on the type and extent of injury, the response may also lead to the development of mature vimentin+ α-smooth muscle actin+ desmin+ myofibroblasts. Myofibroblasts are specialized fibroblastic cells generated in the cornea from keratocyte-derived or bone marrow-derived precursor cells. The disorganized extracellular matrix components secreted by myofibroblasts, in addition to decreased expression of corneal crystallins in these cells, are central biological processes that result in corneal stromal fibrosis associated with opacity or "haze". Several factors are associated with myofibroblast generation and haze development after PRK surgery in rabbits, a reproducible model of scarring, including the amount of tissue ablated, which may relate to the extent of keratocyte apoptosis in the early response to injury, irregularity of stromal surface after surgery, and changes in corneal stromal proteoglycans, but normal regeneration of the epithelial basement membrane (EBM) appears to be a critical factor determining whether a cornea heals with relative transparency or vision-limiting stromal opacity. Structural and functional abnormalities of the regenerated EBM facilitate prolonged entry of epithelium-derived growth factors such as transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) into the stroma that both drive development of mature myofibroblasts from precursor cells and lead to persistence of the cells in the anterior stroma. A major discovery that has contributed to our understanding of haze development is that keratocytes and corneal

  19. Sub-micron and nanoscale feature depth modulates alignment of stromal fibroblasts and corneal epithelial cells in serum-rich and serum-free media.

    Science.gov (United States)

    Fraser, Sarah A; Ting, Yuk-Hong; Mallon, Kelly S; Wendt, Amy E; Murphy, Christopher J; Nealey, Paul F

    2008-09-01

    Topographic features are generally accepted as being capable of modulating cell alignment. Of particular interest is the potential that topographic feature geometry induces cell alignment indirectly through impacting adsorbed proteins from the cell culture medium on the surface of the substrate. However, it has also been reported that micron-scale feature depth significantly impacts the level of alignment of cellular populations on topography, despite being orders of magnitude larger than the average adsorbed protein layer (nm). In order to better determine the impact of biomimetic length scale topography and adsorbed protein interaction on cellular morphology we have systematically investigated the effect of combinations of sub-micron to nanoscale feature depth and lateral pitch on corneal epithelial cell alignment. In addition we have used the unique properties of a serum-free media alternative in direct comparison to serum-rich medium to investigate the role of culture medium protein composition on cellular alignment to topographically patterned surfaces. Our observation that increasing groove depth elicited larger populations of corneal epithelial cells to align regardless of culture medium composition and of cell orientation with respect to the topography, suggests that these cells can sense changes in topographic feature depths independent of adsorbed proteins localized along ridge edges and tops. However, our data also suggests a strong combinatory effect of topography with culture medium composition, and also a cell type dependency in determining the level of cell elongation and alignment to nanoscale topographic features.

  20. Activation of focal adhesion kinase enhances the adhesion of Fusarium solani to human corneal epithelial cells via the tyrosine-specific protein kinase signaling pathway.

    Science.gov (United States)

    Pan, Xiaojing; Wang, Ye; Zhou, Qingjun; Chen, Peng; Xu, Yuanyuan; Chen, Hao; Xie, Lixin

    2011-03-05

    To determine the role of the integrin-FAK signaling pathway triggered by the adherence of F. solani to human corneal epithelial cells (HCECs). After pretreatment with/without genistein, HCECs were incubated with F. solani spores at different times (0-24 h). Cell adhesion assays were performed by optical microscopy. Changes of the ultrastructure were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of F-actin and Paxillin (PAX) were detected by immunofluorescence and western blotting to detect the expression of these key proteins with/without genistein treatment. Cell adhesion assays showed that the number of adhered spores began to rise at 6 h after incubation and peaked at 8 h. SEM and TEM showed that the HCECs exhibited a marked morphological alteration induced by the attachment and entry of the spores. The expression of PAX increased, while the expression of F-actin decreased by stimulation with F. solani. The interaction of F. solani with HCECs causes actin rearrangement in HCECs. Genistein strongly inhibited FAK phosphorylation and the activation of the downstream protein (PAX). F. solani-induced enhancement of cell adhesion ability was inhibited along with the inhibition of FAK phosphorylation. Our results suggest that the integrin-FAK signaling pathway is involved in the control of F. solani adhesion to HCECs and that the activation of focal adhesion kinase enhances the adhesion of human corneal epithelial cells to F. solani via the tyrosine-specific protein kinase signaling pathway.

  1. Safety of Cultivated Limbal Epithelial Stem Cell Transplantation for Human Corneal Regeneration

    Directory of Open Access Journals (Sweden)

    J. Behaegel

    2017-01-01

    Full Text Available Ex vivo cultivated limbal stem cell transplantation is a promising technique for the treatment of limbal stem cell deficiency. While the results of the clinical trials have been extensively reported since the introduction of the technique in 1997, little has been reported regarding the potential health risks associated with production processes and transplantation techniques. Culture procedures require the use of animal and/or human-derived products, which carry the potential of introducing toxic or infectious agents through contamination with known or unknown additives. Protocols vary widely, and the risks depend on the local institutional methods. Good manufacturing practice and xeno-free culture protocols could reduce potential health risks but are not yet a common practice worldwide. In this review, we focus on the safety of both autologous- and allogeneic-cultivated limbal stem cell transplantation, with respect to culture processes, surgical approaches, and postoperative strategies.

  2. Activation of cytokines and NF-kappa B in corneal epithelial cells infected by respiratory syncytial virus: potential relevance in ocular inflammation and respiratory infection

    Directory of Open Access Journals (Sweden)

    Oakes John E

    2004-07-01

    Full Text Available Abstract Background Respiratory syncytial virus (RSV is a major cause of lower respiratory tract infection, claiming millions of lives annually. The virus infects various cells of the respiratory tract as well as resident inflammatory cells such as macrophages. Infection activates a variety of cellular factors such as cytokines and the pro-inflammatory transcription factor, NF-kappa B, all of which are important players in the respiratory disease. However, the exact natural route of RSV infection and its etiology remain relatively unknown. In this paper, we test the hypothesis that human corneal epithelial cells, which constitute the outermost layer of the cornea, can be infected with RSV, and that the infection leads to the activation of proinflammatory macromolecules. Results Corneal swabs obtained from pediatric patients with acute respiratory disease were found to contain RSV at a high frequency (43 positive out of 72 samples, i.e., 60%. Primary corneal epithelial cells in tissue culture supported robust infection and productive growth of RSV. Infection resulted in the activation of TNF-α, IL-6 and sixteen chemokines as well as NF-κB. Three proinflammatory CXC chemokines (MIG, I-TAC, IP-10 underwent the greatest activation. Conclusions The ocular epithelium is readily infected by RSV. The pro-inflammatory cytokines are likely to play critical roles in the etiology of inflammation and conjunctivitis commonly seen in pediatric patients with respiratory infections. RSV-eye interactions have important implications in RSV transmission, immunopathology of RSV disease, and in the management of conjunctivitis.

  3. Characterization of enhancers and the role of the transcription factor KLF7 in regulating corneal epithelial differentiation.

    Science.gov (United States)

    Klein, Rachel Herndon; Hu, William; Kashgari, Ghaidaa; Lin, Ziguang; Nguyen, Tuyen; Doan, Michael; Andersen, Bogi

    2017-09-15

    During tissue development, transcription factors bind regulatory DNA regions called enhancers, often located at great distances from the genes they regulate, to control gene expression. The enhancer landscape during embryonic stem cell differentiation has been well characterized. By contrast, little is known about the shared and unique enhancer regulatory mechanisms in different ectodermally derived epithelial cells. Here, we use ChIP-seq to identify domains enriched for histone marks H3K4me3, H3K4me1, and H3K27ac, and define for the first time the super enhancers and typical enhancers active in primary human corneal epithelial cells. We show that regulatory regions are often shared between cell types of the ectodermal lineage and that corneal epithelial super enhancers are already marked as potential regulatory domains in embryonic stem cells. Kruppel-like factor (KLF) motifs were enriched in corneal epithelial enhancers, consistent with the important roles of KLF4 and KLF5 in promoting corneal epithelial differentiation. We now show that the Kruppel family member KLF7 promotes the corneal progenitor cell state: on many genes, KLF7 antagonized the corneal differentiation-promoting KLF4. Furthermore, we found that two SNPs previously linked to corneal diseases, astigmatism and Stevens-Johnson syndrome, fall within corneal epithelial enhancers and alter their activity by disrupting transcription factor motifs that overlap these SNPs. Taken together, our work defines regulatory enhancers in corneal epithelial cells, highlights global gene-regulatory relationships shared among different epithelial cells, identifies a role for KLF7 as a KLF4 antagonist in corneal epithelial cell differentiation, and explains how two SNPs may contribute to corneal diseases. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  4. Persistent corneal epithelial defect responding to rebamipide ophthalmic solution in a patient with diabetes

    Directory of Open Access Journals (Sweden)

    Hayashi Y

    2016-05-01

    Full Text Available Yusuke Hayashi, Hiroshi Toshida, Yusuke Matsuzaki, Asaki Matsui, Toshihiko Ohta Department of Ophthalmology, Juntendo University Shizuoka Hospital, Izunokuni, Shizuoka, Japan Objective: Rebamipide ophthalmic suspension was developed for the treatment of dry eyes and for other corneal diseases, promoting the secretion of both mucin in tear fluid and membrane-associated mucin, increasing the number of goblet cells, and restoring the barrier function of the corneal epithelium. We report a case of a persistent corneal epithelial defect in a patient with diabetes treated with topical application of rebamipide ophthalmic suspension. Case presentation: A 73-year-old woman had a history of type 2 diabetes for 35 years and nonproliferative diabetic retinopathy for 23 years. She presented to our department with discharge and ophthalmalgia in the left eye. A corneal ulcer was detected, and culture of corneal scrapings was performed, with Staphylococcus aureus and Streptococcus canis being isolated. The infection was treated with levofloxacin eye drops and ofloxacin ophthalmic ointment based on the sensitivity profile of the isolate. However, a corneal epithelial defect persisted for approximately 2 months despite continuing treatment with 0.1% hyaluronic acid ophthalmic suspension and 0.3% ofloxacin eye ointment. Her hemoglobin A1c was 7.3%. The persistent corneal epithelial defect showed improvement at 2 weeks after treatment with rebamipide unit dose 2% ophthalmic suspension, and it did not recur even when vitrectomy was subsequently performed for vitreous hemorrhage due to progression of diabetic retinopathy. Conclusion: This is the first report about efficacy of rebamipide unit dose 2% ophthalmic suspension for presenting persistent corneal epithelial defect in a patient with diabetes. In the present case, the suggested mechanisms are the following: improving the corneal barrier function, stabilization of mucin on the keratoconjunctival epithelium, and

  5. Persistent corneal epithelial defect responding to rebamipide ophthalmic solution in a patient with diabetes.

    Science.gov (United States)

    Hayashi, Yusuke; Toshida, Hiroshi; Matsuzaki, Yusuke; Matsui, Asaki; Ohta, Toshihiko

    2016-01-01

    Rebamipide ophthalmic suspension was developed for the treatment of dry eyes and for other corneal diseases, promoting the secretion of both mucin in tear fluid and membrane-associated mucin, increasing the number of goblet cells, and restoring the barrier function of the corneal epithelium. We report a case of a persistent corneal epithelial defect in a patient with diabetes treated with topical application of rebamipide ophthalmic suspension. A 73-year-old woman had a history of type 2 diabetes for 35 years and nonproliferative diabetic retinopathy for 23 years. She presented to our department with discharge and ophthalmalgia in the left eye. A corneal ulcer was detected, and culture of corneal scrapings was performed, with Staphylococcus aureus and Streptococcus canis being isolated. The infection was treated with levofloxacin eye drops and ofloxacin ophthalmic ointment based on the sensitivity profile of the isolate. However, a corneal epithelial defect persisted for approximately 2 months despite continuing treatment with 0.1% hyaluronic acid ophthalmic suspension and 0.3% ofloxacin eye ointment. Her hemoglobin A1c was 7.3%. The persistent corneal epithelial defect showed improvement at 2 weeks after treatment with rebamipide unit dose 2% ophthalmic suspension, and it did not recur even when vitrectomy was subsequently performed for vitreous hemorrhage due to progression of diabetic retinopathy. This is the first report about efficacy of rebamipide unit dose 2% ophthalmic suspension for presenting persistent corneal epithelial defect in a patient with diabetes. In the present case, the suggested mechanisms are the following: improving the corneal barrier function, stabilization of mucin on the keratoconjunctival epithelium, and improving the wettability and stability of the tear film, which resulted in the promotion of healing of the corneal epithelial defect in a short time period.

  6. Experimental study on differentiation of mouse embryonic stem cells into corneal epithelial cells%鼠胚胎干细胞定向诱导为角膜上皮细胞的实验研究

    Institute of Scientific and Technical Information of China (English)

    杨成明; 滕利; 黄谙飞

    2013-01-01

    目的 建立能够稳定表达绿色荧光蛋白(green fluorescent protein,GFP)的鼠胚胎干细胞系,并研究体外诱导鼠胚胎干细胞(embryonic stem cell,Es细胞)向角膜上皮细胞分化的可能性及条件,为角膜损伤提供新型的上皮修复组织来源.方法 质粒pEGFP-N1脂质体复合体转染鼠胚胎干细胞,对稳定表达GFP的ES细胞以Ⅳ型胶原作为诱导条件,定向诱导鼠ES细胞向角膜上皮细胞转化,观察培养后形态改变,免疫荧光及RT-PCR检测K12、K14及CD44的表达.结果 转染后ES细胞稳定表达GFP,成功诱导出角膜上皮样细胞,呈典型上皮样细胞形态,免疫组化及RT-PCR检测显示,K12及CD44表达阳性,K14表达阴性.结论 转染GFP的ES细胞,在Ⅳ型胶原的诱导下,成功向角膜上皮细胞分化.%Objective To establish mouse embryonic stem cell lines which can stably express green fluorescent protein (green fluorescent protein, GFP), and to study the possibility and conditions of differentiation of mouse embryonic stem (ES) cells into corneal epithelial cells in vitro, in order to serve new source of tissues for epithelial repair. Methods Plasmid pEGFP-N1 liposome complexes was used to transfect mouse ES cells and those cells stably expressing GFP were induced into corneal epithelial cells with type Ⅳ collagen as induction conditions. The resulting corneal epithelial cells were cultured and the morphological changes were observed. Immunofluorescence, RT-PCR were used to detect K12, K14 and CD44 expression. Results After transfection, the ES cells stably expressed GFP, and successfully induced to corneal epithelial cells with typical epithelioid cell morphology. Immunohistochemical and RT-PCR detection showed that the expression of K12 and CD44 were positive and the expression of K14 was negative. Conclusion In the induction of type Ⅳ collagen, ES cells transfect with GFP successfully differentiate to corneal epithelial cells.

  7. Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

    Science.gov (United States)

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2014-05-29

    Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist

  8. Active Pedicle Epithelial Flap Transposition Combined with Amniotic Membrane Transplantation for Treatment of Nonhealing Corneal Ulcers

    Directory of Open Access Journals (Sweden)

    Ting Zhang

    2016-01-01

    Full Text Available Introduction. The objective was to evaluate the efficacy of active pedicle epithelial flap transposition combined with amniotic membrane transplantation (AMT in treating nonhealing corneal ulcers. Material and Methods. Eleven patients (11 eyes with nonhealing corneal ulcer who underwent the combined surgery were included. Postoperatively, ulcer healing time was detected by corneal fluorescein staining. Visual acuity, intraocular pressure, surgical complications, and recurrence were recorded. Corneal status was inspected by the laser scanning confocal microscopy and anterior segment optical coherence tomography (AS-OCT. Results. The primary diseases were herpes simplex keratitis (8 eyes, corneal graft ulcer (2 eyes, and Stevens-Johnson syndrome (1 eye. All epithelial flaps were intact following surgery, without shedding or displacement. Mean ulcer healing time was 10.8±3.1 days, with a healing rate of 91%. Vision significantly improved from 1.70 to 0.82 log MAR (P=0.001. A significant decrease in inflammatory cell infiltration and corneal stromal edema was revealed 2 months postoperatively by confocal microscopy and AS-OCT. Corneal ulcer recurred in 1 eye. None of the patients developed major complications. Conclusion. Active pedicle epithelial flap transposition combined with AMT is a simple and effective treatment for nonhealing corneal ulcers.

  9. Regulation of human corneal epithelial mucins by rebamipide.

    Science.gov (United States)

    Itoh, Shinsaku; Itoh, Kuni; Shinohara, Hisashi

    2014-02-01

    Membrane-associated mucins (MAMs) play important roles in barrier function and tear stability, and their expression on the ocular surface is altered in dry eye disease. Rebamipide is a mucin secretagogue that promotes the production of mucin-like glycoproteins in human corneal epithelial (HCE) cells. However, the expression of MAMs on the corneal epithelia (MUC1, MUC4, MUC16), which is induced by rebamipide, is poorly understood. In this study, we investigated the effect of rebamipide on the regulation of MAM expression in HCE cells. MUC16, Ki67 and PCNA expression levels in HCE cells isolated at confluence and at 24 hours after confluence were examined by Western blotting to assess cell proliferation. HCE cells isolated at 24 hours after confluence were cultured in medium supplemented with 1-10 µM rebamipide or 0.3-30 nM of epidermal growth factor (EGF). Real-time PCR (RT-PCR) and Western blot analysis of MAMs were performed to evaluate the effect of rebamipide. Western blot analysis of cells treated with an EGF receptor inhibitor (AG1478) or MEK1/2 inhibitor (U0126) was performed to reveal the relationship between EGF receptor activation and rebamipide-induced MAM expression. HCE cells isolated at 24 hours after confluence had lower cell proliferation activity and increased MUC16 expression compared with cells isolated at confluence. RT-PCR and Western blot analysis revealed that rebamipide increased MAM gene expression for 2 hours and protein expression for 24 hours in HCE cells. EGF inhibitor treatment led to reduced levels of all three MAMs that are normally induced by rebamipide, whereas EGF induced the expression of all three MAMs. We suggested that rebamipide increased MUC1, MUC4 and MUC16 expression levels through signals involved in EGF receptor activation in the human corneal epithelia. These data suggest that rebamipide may improve subjective symptoms of dry eye disease by upregulating MAM expression.

  10. Molecular mechanisms of dust-induced toxicity in human corneal epithelial cells: Water and organic extract of office and house dust.

    Science.gov (United States)

    Xiang, Ping; Liu, Rong-Yan; Sun, Hong-Jie; Han, Yong-He; He, Rui-Wen; Cui, Xin-Yi; Ma, Lena Q

    2016-01-01

    Human corneal epithelial (HCE) cells are continually exposed to dust in the air, which may cause corneal epithelium damage. Both water and organic soluble contaminants in dust may contribute to cytotoxicity in HCE cells, however, the associated toxicity mechanisms are not fully elucidated. In this study, indoor dust from residential houses and commercial offices in Nanjing, China was collected and the effects of organic and water soluble fraction of dust on primary HCE cells were examined. The concentrations of heavy metals in the dust and dust extracts were determined by ICP-MS and PAHs by GC-MS, with office dust having greater concentrations of heavy metals and PAHs than house dust. Based on LC50, organic extract was more toxic than water extract, and office dust was more toxic than house dust. Accordingly, the organic extracts induced more ROS, malondialdehyde, and 8-Hydroxydeoxyguanosine and higher expression of inflammatory mediators (IL-1β, IL-6, and IL-8), and AhR inducible genes (CYP1A1, and CYP1B1) than water extracts (pdust presented greater suppression of superoxide dismutase and catalase activity than those of house dust. In addition, exposure to dust extracts activated NF-κB signal pathway except water extract of house dust. The results suggested that both water and organic soluble fractions of dust caused cytotoxicity, oxidative damage, inflammatory response, and activation of AhR inducible genes, with organic extracts having higher potential to induce adverse effects on primary HCE cells. The results based on primary HCE cells demonstrated the importance of reducing contaminants in indoor dust to reduce their adverse impacts on human eyes.

  11. Cytocompatibility of Three Corneal Cell Types with Amniotic Membrane

    Institute of Scientific and Technical Information of China (English)

    CHENJian-su; CHENRui; XUJin-tang; DINGYong; ZHAOSong-bin; LISui-lian

    2004-01-01

    Rabbit limbal corneal epithelial cells, corneal endothelial cells and keratocytes were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Histological and scan electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. Results showed that three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints which could form networks. In conclusion, amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea.

  12. Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

    Institute of Scientific and Technical Information of China (English)

    GAO Nan; CUI GuangHui; WANG ZhiChong; HUANG Bing; GE Jian; LU Rong; ZHANG KeFei; FAN ZhiGang; LU Li; PENG Zhan

    2007-01-01

    Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epidermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immunohistology and RT-PCR were conducted to identify the expression of specific markers (β1, α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epidermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being cocultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β 1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.

  13. Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epi-dermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immuno-histology and RT-PCR were conducted to identify the expression of specific markers (β1, α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epi-dermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being co-cultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β 1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.

  14. Involvement of YAP, TAZ and HSP90 in contact guidance and intercellular junction formation in corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Vijay Krishna Raghunathan

    Full Text Available The extracellular environment possesses a rich milieu of biophysical and biochemical signaling cues that are simultaneously integrated by cells and influence cellular phenotype. Yes-associated protein (YAP and transcriptional co-activator with PDZ-binding motif (WWTR1; TAZ, two important signaling molecules of the Hippo pathway, have been recently implicated as nuclear relays of cytoskeletal changes mediated by substratum rigidity and topography. These proteins intersect with other important intracellular signaling pathways (e.g. Wnt and TGFβ. In the cornea, epithelial cells adhere to the stroma through a 3-dimensional topography-rich basement membrane, with features in the nano-submicron size-scale that are capable of profoundly modulating a wide range of fundamental cell behaviors. The influences of substratum-topography, YAP/TAZ knockdown, and HSP90 inhibition on cell morphology, YAP/TAZ localization, and the expression of TGFβ2 and CTGF, were investigated. The results demonstrate (a that knockdown of TAZ enhances contact guidance in a YAP dependent manner, (b that CTGF is predominantly regulated by YAP and not TAZ, and (c that TGFβ2 is regulated by both YAP and TAZ in these cells. Additionally, inhibition of HSP90 resulted in nuclear localization and subsequent transcriptional-activation of YAP, formation of cell-cell junctions and co-localization of E-cadherin and β-catenin at adherens junctions. Results presented in this study reflect the complexities underlying the molecular relationships between the cytoskeleton, growth factors, heat shock proteins, and co-activators of transcription that impact mechanotransduction. The data reveal the importance of YAP/TAZ on the cell behaviors, and gene and protein expression.

  15. Effects of diabetic keratopathy on corneal optical density, central corneal thickness, and corneal endothelial cell counts.

    Science.gov (United States)

    Gao, Feng; Lin, Tao; Pan, Yingzhe

    2016-09-01

    Diabetic keratopathy is an ocular complication that occurs with diabetes. In the present study, the effect of diabetic keratopathy on corneal optical density, central corneal thickness, and corneal endothelial cell count was investigated. One hundred and eighty diabetic patients (360 eyes) were enrolled in the study during the period from March, 2012 to March, 2013. The patients were divided into three age groups: 10 years, with 60 patients per group (120 eyes). During the same period, 60 healthy cases (120 eyes) were selected and labeled as the normal control group. The Pentacam was used to measure the corneal optical density, and central corneal thickness. Specular microscopy was used to examine the corneal endothelial cell density. The coefficient of partial correlation was used to control age and correlate the analysis between the corneal optical density, corneal endothelial cell density, and central corneal thickness. The stage of the disease, the medial and intimal corneal optical density and central corneal thickness was analyzed in the diabetes group. The corneal optical density in the diabetes group increased compared with that of the normal control group. The medial and intimal corneal optical density and central corneal thickness were positively correlated with the course of the disease. However, the corneal endothelial cell density was not associated with the course of diabetes. There was a positive association between the medial and intimal corneal optical density and central corneal thickness of the diabetic patients. In conclusion, the results of the present study show that medial and intimal corneal optical density and central corneal thickness were sensitive indicators for early diabetic keratopathy.

  16. Triggering of toll-like receptors 2 and 4 by Aspergillus fumigatus conidia in immortalized human corneal epithelial cells to induce inflammatory cytokines

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jie; WU Xin-yi

    2008-01-01

    Background Cornea epithelial cells play early and crucial roles in the initiation of ocular surface responses to pathogens.Participation of toll-like receptor(TLR)2 and TLR4,which are major forms of fungi receptors,may be involved in Aspergillus fumigatus induced immune responses.The obiective of the present study was to examine whether inactive Aspergillus fumigatus conidia induce NF-κB activation and production of proinflammaory cytokines,and whether the expression of TLR2 and TLR4 were amplified by conidia in cultured immortalized human corneal epithelial cells (THCEs).This may contribute to our knowledge of the mechanism by which the host cornea can successfully defend against invasive fungi.Methods Aspergillus fumigatus conidia were used to challenge THCE cells.THCE cells were harvested after 0.5,1,2or 4 hours incubation.Real-time quantitative PCR was performed to determine the expression of TLR2,TLR4,TNF-α and IL-8.Western blotting was performed to determine the expression of NF-κB.Enzyme-linked immunosorbent assay (EUSA)was performed to determine the expression of TNF-α and IL-8.And the release of TNF-α and IL-8 in the cell supematant were also assessed by ELISA with or without pretreatment with TLR2 and TLR4 neutralizing antibodies.Results Aspergillus fumigatus conidia elicited the expression of TLR2,TLR4,TNF-α and IL-8 mRNA in THCEs.Exposure of THCE cells to Aspergillus fumigatus conidia resulted in NF-κB activation,which increased at 30 minutes (increased from 11.35±2.74 in the controls to 19.12±3.48,P<0.05)and thereafter increased steadily up to 4 hours after challenge(P<0.01).Concomitant with NF-κB acfivation,secretion of TNF-α and IL-8 in conidia-challenged cells was increased in a time-dependent manner.Incubation of THCE cells with TLR2 antibody or TLR4 antibody before conidia challenge resulted in jnhibifion of conidia-induced TNF-α and IL-8 secretion(P<0.05),TLR2 antibody and TLR4 antibody together significantly increased

  17. Advanced glycation end products induce human corneal epithelial cells apoptosis through generation of reactive oxygen species and activation of JNK and p38 MAPK pathways.

    Directory of Open Access Journals (Sweden)

    Long Shi

    Full Text Available Advanced Glycation End Products (AGEs has been implicated in the progression of diabetic keratopathy. However, details regarding their function are not well understood. In the present study, we investigated the effects of intracellular reactive oxygen species (ROS and JNK, p38 MAPK on AGE-modified bovine serum albumin (BSA induced Human telomerase-immortalized corneal epithelial cells (HUCLs apoptosis. We found that AGE-BSA induced HUCLs apoptosis and increased Bax protein expression, decreased Bcl-2 protein expression. AGE-BSA also induced the expression of receptor for advanced glycation end product (RAGE. AGE-BSA-RAGE interaction induced intracellular ROS generation through activated NADPH oxidase and increased the phosphorylation of p47phox. AGE-BSA induced HUCLs apoptosis was inhibited by pretreatment with NADPH oxidase inhibitors, ROS quencher N-acetylcysteine (NAC or neutralizing anti-RAGE antibodies. We also found that AGE-BSA induced JNK and p38 MAPK phosphorylation. JNK and p38 MAPK inhibitor effectively blocked AGE-BSA-induced HUCLs apoptosis. In addition, NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA. Our results indicate that AGE-BSA induced HUCLs apoptosis through generation of intracellular ROS and activation of JNK and p38 MAPK pathways.

  18. Corneal stem cells and tissue engineering: Current advancesand future perspectives

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Major advances are currently being made in regenerativemedicine for cornea. Stem cell-based therapiesrepresent a novel strategy that may substituteconventional corneal transplantation, albeit there aremany challenges ahead given the singularities of eachcellular layer of the cornea. This review recapitulatesthe current data on corneal epithelial stem cells,corneal stromal stem cells and corneal endothelialcell progenitors. Corneal limbal autografts containingepithelial stem cells have been transplanted in humansfor more than 20 years with great successful rates,and researchers now focus on ex vivo cultures andother cell lineages to transplant to the ocular surface.A small population of cells in the corneal endotheliumwas recently reported to have self-renewal capacity,although they do not proliferate in vivo . Two mainobstacles have hindered endothelial cell transplantationto date culture protocols and cell delivery methods tothe posterior cornea in vivo . Human corneal stromalstem cells have been identified shortly after therecognition of precursors of endothelial cells. Stromalstem cells may have the potential to provide a directcell-based therapeutic approach when injected tocorneal scars. Furthermore, they exhibit the ability todeposit organized connective tissue in vitro and maybe useful in corneal stroma engineering in the future.Recent advances and future perspectives in the field arediscussed.

  19. Corneal Epithelial Remodeling and Its Effect on Corneal Asphericity after Transepithelial Photorefractive Keratectomy for Myopia

    Directory of Open Access Journals (Sweden)

    Jie Hou

    2016-01-01

    Full Text Available Purpose. To evaluate the changes in epithelial thickness profile following transepithelial photorefractive keratectomy (T-PRK for myopia and to investigate the effect of epithelial remodeling on corneal asphericity. Methods. Forty-four patients (44 right eyes who underwent T-PRK were retrospectively evaluated. Epithelial thickness was measured using spectral-domain optical coherence tomography at different corneal zones (central, 2 mm; paracentral, 2–5 mm; and mid-peripheral, 5-6 mm preoperatively and at 1 week and 1, 3, and 6 months postoperatively. The correlation between the changes in corneal epithelial thickness (ΔCET and postoperative Q-value changes (ΔQ was analyzed 6 months postoperatively. Results. Epithelial thickness at 6 months showed a negative meniscus-like lenticular pattern with less central thickening, which increased progressively toward the mid-periphery (3.69±4.2, 5.19±3.8, and 6.23±3.9 μm at the center, paracenter, and mid-periphery, resp., P<0.01. A significant positive relationship was observed between epithelial thickening and ΔQ 6 months postoperatively (r=0.438, 0.580, and 0.504, resp., P<0.01. Conclusions. Significant epithelial thickening was observed after T-PRK and showed a lenticular change with more thickening mid-peripherally, resulting in increased oblateness postoperatively. Epithelial remodeling may modify the epithelial thickness profile after surface ablation refractive surgery for myopia.

  20. F 2 excimer laser (157 nm) radiation modification and surface ablation of PHEMA hydrogels and the effects on bioactivity: Surface attachment and proliferation of human corneal epithelial cells

    Science.gov (United States)

    Zainuddin; Chirila, Traian V.; Barnard, Zeke; Watson, Gregory S.; Toh, Chiong; Blakey, Idriss; Whittaker, Andrew K.; Hill, David J. T.

    2011-02-01

    Physical and chemical changes at the surface of poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels modified by ablation with an F 2 excimer laser were investigated experimentally. An important observation was that only the outer exposed surface layers of the hydrogel were affected by the exposure to 157 nm radiation. The effect of the surface changes on the tendency of cells to adhere to the PHEMA was also investigated. A 0.5 cm 2 area of the hydrogel surfaces was exposed to laser irradiation at 157 nm to fluences of 0.8 and 4 J cm -2. The changes in surface topography were analysed by light microscopy and atomic force microscopy, while the surface chemistry was characterized by attenuated total reflection infrared and X-ray photoelectron spectroscopies. Cell-interfacial interactions were examined based on the proliferation of human corneal limbal epithelial (HLE) cells cultured on the laser-modified hydrogels, and on the unexposed hydrogels and tissue culture plastic for comparison. It was observed that the surface topography of laser-exposed hydrogels showed rippled patterns with a surface roughness increasing at the higher exposure dose. The changes in surface chemistry were affected not only by an indirect effect of hydrogen and hydroxyl radicals, formed by water photolysis, on the PHEMA, but also by the direct action of laser radiation on PHEMA if the surface layers of the gel become depleted of water. The laser treatment led to a change in the surface characteristics, with a lower concentration of ester side-chains and the formation of new oxygenated species at the surface. The surface also became more hydrophobic. Most importantly, the surface chemistry and the newly created surface topographical features were able to improve the attachment, spreading and growth of HLE cells.

  1. Evaluation of inhibitory potential of some selective methanolic plants extracts on biological characteristics of Acanthamoeba castellanii using human corneal epithelial cells in vitro.

    Science.gov (United States)

    Shoaib, Hafiz Muhammad; Muazzam, Ambreen Gul; Mir, Asif; Jung, Suk-Yul; Matin, Abdul

    2013-03-01

    Acanthamoeba is an opportunistic protozoan pathogen and known to be one of the most ubiquitous organisms, play a vital role in ecosystem, and recognized to cause blinding keratitis and rare but fatal granulomatous encephalitis involving the central nervous system with a very poor prognosis. This is due to limited availability of effective anti-Acanthamoeba drugs. The objective of the present study was to determine the efficacy of methanolic plants crude extracts on the viability and biological properties of Acanthamoeba castellanii (T4 genotype) and its cytotoxic effects on human corneal epithelial cells (HCEC). Using HCEC, it was observed that Acanthamoeba exhibited binding (>90 %) and cytotoxicity (>80 %) to host cells. However, plant crude extracts remarkably inhibited more than 70 and 60 % of Acanthamoeba binding and cytotoxicity to HCEC, respectively. It was further established that crude extracts (ranging from 0.1 to 1.5 mg/ml) exhibited amoebicidal effects, i.e., >50 % of trophozoites were killed/reduced at maximum dose (1.5 mg/ml) within 1 h incubation. However, the residual subpopulation remained static over longer incubations. Furthermore, growth assay demonstrated crude extracts inhibited >50 % Acanthamoeba numbers up to 7 days. Our results confirmed that plant crude extracts has inhibitory effects on Acanthamoeba growth and viability. Overall, these findings revealed that tested plant extracts is inhibitory to Acanthamoeba properties associated with pathogenesis. To the best of our knowledge, our findings demonstrated for the first time that selected methanol plant crude extracts exhibits inhibitory effects on biological properties of Acanthamoeba without any toxic effects on HCEC cells in vitro.

  2. Corneal Molecular and Cellular Biology for the Refractive Surgeon: The Critical Role of the Epithelial Basement Membrane.

    Science.gov (United States)

    Marino, Gustavo K; Santhiago, Marcony R; Torricelli, Andre A M; Santhanam, Abirami; Wilson, Steven E

    2016-02-01

    To provide an overview of the recent advances concerning the corneal molecular and cellular biology processes involved in the wound healing response after excimer laser surface ablation and LASIK surgery. Literature review. The corneal wound healing response is a complex cascade of events that impacts the predictability and stability of keratorefractive surgical procedures such as photorefractive keratectomy and LASIK. The generation and persistence of corneal myofibroblasts (contractile cells with reduced transparency) arise from the interaction of cytokines and growth factors such as transforming growth factor beta and interleukin 1 produced by epithelial and stromal cells in response to the corneal injury. Myofibroblasts, and the opaque extracellular matrix they secrete into the stroma, disturb the precise distribution and spacing of collagen fibers related to corneal transparency and lead to the development of vision-limiting corneal opacity (haze). The intact epithelial basement membrane has a pivotal role as a structure that regulates corneal epithelial-stromal interactions. Thus, defective regeneration of the epithelial basement membrane after surgery, trauma, or infection leads to the development of stromal haze. The apoptotic process following laser stromal ablation, which is proportional to the level of attempted correction, leads to an early decrease in anterior keratocyte density and the diminished contribution of these non-epithelial cells of components such as perlecan and nidogen-2 required for normal regeneration of the epithelial basement membrane. Haze persists until late repair of the defective epithelial basement membrane. Defective regeneration of the epithelial basement membrane has a critical role in determining whether a cornea heals with late haze after photorefractive keratectomy or with scarring at the flap edge in LASIK. Copyright 2016, SLACK Incorporated.

  3. The role of electrical signals in murine corneal wound re-epithelialization.

    Science.gov (United States)

    Kucerova, Romana; Walczysko, Petr; Reid, Brian; Ou, Jingxing; Leiper, Lucy J; Rajnicek, Ann M; McCaig, Colin D; Zhao, Min; Collinson, J Martin

    2011-06-01

    Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.

  4. Mechanism of induction of fibroblast to corneal endothelial cell.

    Science.gov (United States)

    Jiang, Yan; Fu, Wei-Cai; Zhang, Lin

    2014-08-01

    To explore mechanism of nduction of fibroblast to corneal endothelial cell. Rabbit conjunctiva fibroblasts were used as feeder cells, rabbit oral mucosa epithelial cells were used as seed cells, and human denuded amniotic membrane was used as carrier to establish tissue engineering corneal endothelium. The transformation effect was observed. As concentration of mitomycin C increased, cell survival rate gradually decreased, cell proliferation was obviously inhibited when concentration≥25 μg/mL; 5 days after being treated by 5 μg/mL mitomycin C, cell body was enlarged and extended without cell fusion, however after being treated by 0.5 μg/mL mitomycin C, cell body was significantly proliferated and gradually fused; after 3 weeks of culture, stratified epithelium appeared on rabbit oral mucosa epithelial cells, differentiation layers were 4-5 and were well differentiated, the morphology was similar to corneal endothelial cells; Under electron microscope, surface layer of cells were polygonal, tightly connected to another with microvilli on the border, there was hemidesmosome between basal cells and human denuded amniotic membrane. Fibroblast cells have the potential of multi-directional differentiation, effective induction can promote emergence of intercellular desmosomes between seed cells and emergence of epithelial surface microvilli, and differentiate to the corneal endothelial cell. However, clinical application still needs more research and safety evaluation. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  5. Epidermal growth factor (EGF)-induced corneal epithelial wound healing through nuclear factor κB subtype-regulated CCCTC binding factor (CTCF) activation.

    Science.gov (United States)

    Wang, Ling; Wu, Xiaolin; Shi, Ting; Lu, Luo

    2013-08-23

    Epidermal growth factor (EGF) plays an important role in corneal epithelial migration and proliferation to improve the wound healing process. This study aimed to understand the role of NFκB in EGF-induced corneal epithelial wound healing through regulation of CTCF activity, which plays important roles in cell motility and migration to promote wound healing. The effect of NFκB p50 on corneal epithelial wound healing was investigated by comparing the eyes of wild-type and p50 knockout mice. We found that there was a significant retardation in corneal epithelial wound healing in the corneas of p50 knockout mice. Wound closure rates were measured in human corneal epithelial cells transfected with an NFκB activation-sensitive CTCF expression construct to demonstrate the effect of human CTCF expression under the control of EGF-induced NFκB activation on wound healing. EGF stimulation activated NFκB, which directly triggered the expression of the exogenous human CTCF in transfected cells and, subsequently, promoted human corneal epithelial cell motility, migration, and wound healing. Overexpression of CTCF in corneal epithelial cells and mouse corneas significantly enhanced the wound healing process. Furthermore, the effect of overexpressing NFκB p50 in corneal epithelial cells on the promotion of wound healing was abolished by knockdown of CTCF with CTCF-specific shRNA. Thus, a direct regulatory relationship between EGF-induced NFκB p50 and CTCF activation affecting corneal epithelial wound healing has been established, indicating that CTCF is, indeed, a NFκB p50-targeted and effective gene product in the core transcriptional network downstream from the growth factor-induced NFκB signaling pathway.

  6. Assessment of corneal epithelial integrity after acute exposure to ocular hypotensive agents preserved with and without benzalkonium chloride.

    Science.gov (United States)

    Whitson, Jess T; Cavanagh, H Dwight; Lakshman, Neema; Petroll, W Matthew

    2006-01-01

    The corneal toxicity of 2 intraocular pressure-lowering agents was compared in a rabbit cornea model with New Zealand White rabbits. Corneal epithelial morphology and cell size were assessed by in vivo confocal microscopy. Baseline microscopic examinations were performed on 1 eye of each animal. Two weeks later, the eyes were bathed for 3 min in travoprost 0.004% preserved without benzalkonium chloride (BAK( or latanoprost 0.005% preserved with 0.02% BAK; the eyes were then rinsed with balanced salt solution, and the corneas were again examined by confocal microscopy (n=4/group). A second group of animals was exposed to the medications through a dosing regimen of 1 drop/min (lpar3 drops total) (n=4/group). In eyes treated with travoprost without BAK (3-min bath), superficial epithelial cells were similar to baseline, as indicated by their visible cell borders and bright nuclei. In contrast, the surface cells in eyes treated with latanoprost were significantly smaller and brighter and had less distinct borders. Surface cell size was significantly smaller as compared with baseline size and as compared with rabbits treated with travoprost without BAK for 3 min. Similar effects on corneal epithelial cell morphology were observed with the 1-drop/min dosing regimen. In this rabbit model, travoprost 0.004% preserved without BAK did not cause corneal epithelial toxicity; latanoprost 0.005% induced superficial cell loss, most likely caused by the presence of a relatively high concentration of BAK (0.02%).

  7. Efficacy of highly hydrophilic soft contact lenses for persistent corneal epithelial defects after anterior segment surgery

    Directory of Open Access Journals (Sweden)

    Zhi-Wei Peng

    2015-02-01

    Full Text Available AIM:To investigate the efficacy of highly hydrophilic soft contact lenses for persistent corneal epithelial defects.METHODS:In this retrospective case analysis, 28 patients(28 eyeswith persistent corneal epithelial defects after anterior segment surgery from January 2011 to June 2013 in our hospital were reviewed. After regular treatment for at least 2wk, the persistent corneal epithelial defects were treated with highly hydrophilic soft contact lenses, until the corneal epithelial healing. Continued to wear the same lens no more than 3wk, or in need of replacement the new one. All cases were followed up for 6mo. Key indicators of corneal epithelial healling, corneal fluorescein staining and ocular symptoms improvement were observed.RESULTS: Twenty-one eyes were cured(75.00%, markedly effective in 5 eyes(17.86%, effective in 2 eyes(7.14%, no invalid cases, the total efficiency of 100.00%. Ocular symptoms of 25 cases(89.29%relieved within 2d, the rest 3 cases(10.71%relieved within 1wk. The corneal epithelial of 6 cases(21.43%repaired in 3wk, 13 cases(46.43%in 6wk, 7 cases(25.00%in 9wk, 2 cases(7.14%over 12wk. There were no signs of secondary infection. And no evidence of recurrence in 6mo. CONCLUSION: Highly hydrophilic soft contact lenses could repair persistent corneal epithelial defects after anterior segment surgery significantly, while quickly and effectively relieve a variety of ocular irritation.

  8. 重视白内障术后角膜上皮细胞功能障碍%Pay attention to the corneal epithelial cell dysfunction after cataract surgery

    Institute of Scientific and Technical Information of China (English)

    孙旭光; 王森

    2015-01-01

    Corneal epithelial dysfunction (CED) is the abnormality of the regeneration,conjunction,adhesion and immigration of the corneal epithelium cells without the decompensation of the corneal limbal cells.Due to the affection resulting from the systemic problems of patients and the management in the preoperative period,some of the patients at one to two weeks after cataraci surgery will present the edema and fluorescein staining of the corneal epithelium.Without correct therapy,the defect of the epithelium,or even persisting ulceration of the cornea will occur.The key points of the management for CED are the early diagnosis and reasonable therapy.We suggest paying special attention to CED in the patients with metabolism diseases,abnormality of the tear film and long-term blepharitis.%角膜上皮细胞功能障碍是指在无角膜缘细胞功能失代偿的条件下,角膜上皮细胞再生、连接、黏附及移行功能异常.由于患者自身及围手术期各种因素的影响,少数白内障患者在术后1~2周,开始出现角膜上皮水肿及角膜点状染色等,在处理不正确时,角膜上皮会发生缺损,甚至发展为迁延性溃疡.治疗的关键在于早期发现与及时正确地处理.对于有全身代谢性疾病、术前泪液异常及长期睑缘炎等情况的患者,应特别注意术后角膜上皮细胞功能障碍.

  9. Aloe vera gel facilitates re-epithelialization of corneal alkali burn in normal and diabetic rats

    Directory of Open Access Journals (Sweden)

    Atiba A

    2015-10-01

    Full Text Available Ayman Atiba,1 Tamer Wasfy,2 Walied Abdo,3 Ahmed Ghoneim,2 Tarek Kamal,4 Mustafa Shukry5 1Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, 2Department of Ophthalmology, Faculty of Medicine, Tanta University, 3Department of Pathology, 4Department of Biochemistry, 5Department of Physiology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt Purpose: To investigate the efficacy of topical applied aloe vera (AV and to facilitate the repair of the standardized alkaline corneal ulcer in normal and diabetic rats.Materials and methods: The corneal alkali-burn injury model was established unilaterally in Wistar rats by filter paper saturated with 0.01 M NaOH contacting the eyes for 45 seconds. Rats were divided into four groups: normal control (NC, normal AV (NAV, diabetic control (DC, and diabetic AV (DAV. NAV and DAV groups were treated with AV gel eye drops four times daily, and NC and DC groups were treated with normal saline for 3 days. Corneal epithelial wound closure and degree of edema were recorded using slit lamp and optical coherence tomography at 0, 24, 48, and 72 hours postwounding. Histological examination was conducted to evaluate the degree of inflammation and the healing effect.Results: Corneal epithelial wound healing was better in the NAV group than in the NC group, and it was significantly higher in the DAV group than in the DC group (P<0.05. In comparison to the DC group, DAV treated with AV demonstrated a marked reduction in edema at 48 and 72 hours. Histologically, corneal re-epithelialization was complete and higher in DAV group than that in DC group; moreover, the inflammatory cells were increased in DC group than DAV group (P<0.05.Conclusion: This study demonstrated the efficacy of AV for enhanced corneal re-epithelialization, as well as reduced inflammatory response after alkali burn in rats; therefore, it could be useful as a

  10. Studies on Toxicity of Combination of Borneol and Benzalkonium Chloride to Red Blood Cells and Corneal Epithelial Cells in Vitro%冰片与苯扎氯铵合用对红细胞及角膜上皮细胞的损伤效应

    Institute of Scientific and Technical Information of China (English)

    廖南英; 伍海涛; 梁小雯; 刘启德; 宓穗卿; 王宁生

    2014-01-01

    目的:考察冰片与苯扎氯铵合用对红细胞及角膜上皮细胞的毒性。方法实验分为溶剂对照组、冰片组、苯扎氯铵组、冰片+苯扎氯铵组,分别测定各组红细胞半数溶血浓度(HC50)和角膜上皮细胞半数抑制浓度(IC50),根据等辐射分析法和响应面分析法判断联合作用类型和趋势,并观察二者合用对眼角膜上皮细胞增殖的影响。结果苯扎氯铵对红细胞及角膜上皮细胞的毒性作用呈明显的量效关系,加入冰片后能降低其HC50和IC50值。结论冰片与苯扎氯铵对红细胞的溶血效应表现为协同作用,冰片能增强苯扎氯铵对角膜上皮细胞的毒性作用,受损一定程度内的细胞可恢复。%Objective The in vitro hemolytic test and cytotoxicity test were used to study the toxicity of the combination of borneol and benzalkonium chloride to red blood cells and corneal epithelial cells. Methods Suspension of rabbit red blood cells and corneal epithelial cells were treated with different concentrations of borneol , benzalkonium chloride and combination of borneol-benzalkonium. Dose-response assays were firstly carried out to obtain the 50%hemolysis concentration(HC50) of the individual drug group. Then isobolographic analysis and response surface analysis were used to define the type and tendency of interaction between borneol and benzalkonium chloride. The effects of the combination on the proliferation of corneal epithelial cells were also observed. Results The toxicity of benzalkonium chloride to red blood cells and corneal epithelial cells was dose-dependent. The curve of dose-effect showed that HC 50 and IC50 of benzalkonium chloride were decreased after borneol was added. Conclusion Borneol and benzalkonium chloride have synergistic action on the hemolytic effect, and borneol can enhance the toxicity of benzalkonium chloride on corneal epithelial cells. The corneal epithelial cells with certain degrees of

  11. A novel collagen film with micro-rough surface structure for corneal epithelial repair fabricated by freeze drying technique

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yang [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Ren, Li, E-mail: psliren@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Wang, Yingjun, E-mail: imwangyj@163.com [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China)

    2014-05-01

    Highlights: • Collagen film with micro-rough surface is fabricated by freeze drying technique. • The film has suitable water uptake capability and toughness performance. • The film has good optical performance. • Human corneal epithelial cells studies confirmed the biocompatibility of the film. - Abstract: Corneal epithelial defect is a common disease and keratoplasty is a common treatment method. A collagen film with micro-rough surface was fabricated through a simple freeze drying technique in this study. Compared with the air-dried collagen film (AD-Col), this freeze-dried collagen film (FD-Col) has a more suitable water uptake capability (about 85.5%) and toughness performance. Both of the two films have good optical properties and the luminousness of them is higher than 80%. Besides, the adhesion and proliferation rate of human corneal epithelial cells on the micro-rough surface of FD-Col film is higher than that on the smooth surface of AD-Col film. The results indicate that this FD-Col film may have potential applications for corneal epithelial repair.

  12. Iontophoresis-Assisted Corneal Collagen Cross-Linking with Epithelial Debridement: Preliminary Results

    Science.gov (United States)

    Legrottaglie, Emanuela F.

    2016-01-01

    Purpose. To report the early outcomes of iontophoresis-assisted corneal collagen cross-linking procedure with epithelial debridement (I-SCXL). Methods. Twenty eyes of twenty patients with progressive keratoconus were included in this prospective clinical study. Best spectacle corrected visual acuity (BSCVA), sphere and cylinder refraction, corneal topography, Scheimpflug tomography, aberrometry, anterior segment optical coherence tomography (AS-OCT), and endothelial cell count were assessed at baseline and at 1, 3, and 6 months of follow-up. The parameters considered to establish keratoconus progression were always proven with differential maps as change in curvature in the cone area of at least 1 diopter obtained with an instantaneous map. Results. Functional parameters showed a significant improvement (p 0.05). Conclusion. The early results indicate that the I-SCXL may be able to reduce the treatment time and improve the riboflavin diffusion. PMID:27547758

  13. Iontophoresis-Assisted Corneal Collagen Cross-Linking with Epithelial Debridement: Preliminary Results

    Directory of Open Access Journals (Sweden)

    Paolo Vinciguerra

    2016-01-01

    Full Text Available Purpose. To report the early outcomes of iontophoresis-assisted corneal collagen cross-linking procedure with epithelial debridement (I-SCXL. Methods. Twenty eyes of twenty patients with progressive keratoconus were included in this prospective clinical study. Best spectacle corrected visual acuity (BSCVA, sphere and cylinder refraction, corneal topography, Scheimpflug tomography, aberrometry, anterior segment optical coherence tomography (AS-OCT, and endothelial cell count were assessed at baseline and at 1, 3, and 6 months of follow-up. The parameters considered to establish keratoconus progression were always proven with differential maps as change in curvature in the cone area of at least 1 diopter obtained with an instantaneous map. Results. Functional parameters showed a significant improvement (p0.05. Conclusion. The early results indicate that the I-SCXL may be able to reduce the treatment time and improve the riboflavin diffusion.

  14. Nerve Growth Factor Promotes Corneal Epithelial Migration by Enhancing Expression of Matrix Metalloprotease-9

    Science.gov (United States)

    Blanco-Mezquita, Tomas; Martinez-Garcia, Carmen; Proença, Rui; Zieske, James D.; Bonini, Stefano; Lambiase, Alessandro; Merayo-Lloves, Jesus

    2013-01-01

    Purpose. Nerve growth factor (NGF) is a neuropeptide essential for the development, survival, growth, and differentiation of corneal cells. Its effects are mediated by both TrkA and p75 receptors. Clinically relevant use of NGF was introduced to treat neurotrophic ulcerations in patients. Herein, we examine the mechanisms by which NGF enhances epithelial wound healing both in vivo and in vitro. Methods. An animal model using adult hens was implemented for the in vivo experiments. Laser ablation keratectomy was performed and animals were observed for up to 7 days. Epithelial healing was measured with fluorescein. In addition, proliferation was measured using BrdU incorporation and both TrkA and matrix metalloprotease-9 (MMP-9) expression were measured by immunohistochemistry (IHC) and Western blot (WB). In vitro experiments were carried out with telomerase-immortalized human corneal epithelial cells (HCLE). The rate of proliferation was measured using a colorimetric assay and BrdU incorporation. Real-time migration was evaluated with an inverted microscope. MMP-9 expression was evaluated by immunocytochemistry (ICC), WB, zymography, and RT-PCR. Finally, beta-4 integrin (β4) expression was assessed by ICC and WB. Results. Faster epithelial healing was observed in NGF-treated corneas compared with controls (P < 0.01). These corneas showed increased proliferation, TrkA upregulation, and enhanced MMP-9 presence (P < 0.01). In vitro, faster spreading and migration were observed in response to NGF (P < 0.01). Enhanced proliferation, as well as enhanced TrkA and MMP-9 expression, and decreased β4 levels were observed after adding NGF (P < 0.01). Conclusions. NGF plays a major role during the epithelial healing process by promoting migration, a process that is accelerated by cell spreading. This effect is mediated by both the upregulation of MMP-9 and cleavage of β4 integrin. PMID:23640040

  15. 1,25(OH)2D3 and VDR Signaling Pathways Regulate the Inhibition of Dectin-1 Caused by Cyclosporine A in Response to Aspergillus Fumigatus in Human Corneal Epithelial Cells

    Science.gov (United States)

    Xia, Yiping; Zhao, Guiqiu; Lin, Jing; Li, Cui; Cong, Lin; Jiang, Nan; Xu, Qiang; Wang, Qian

    2016-01-01

    Background The objective of this study is to observe whether cyclosporine A (CsA) inhibits the expression of dectin-1 in human corneal epithelial cells infected with Aspergillus fumigatus (A. fumigatus) and to investigate the molecular mechanisms of the inhibition. Methods Immortalized human corneal epithelial cells (HCECs) were pretreated with 1,25(OH)2D3 and VDR inhibitor for 1 h, and then they were pretreated with CsA for 12h. After these pretreatments, the HCECs were stimulated with A. fumigatus and curdlan respectively, and the expression of dectin-1 and proinflammatory cytokines (IL-1β and TNF-α) were detected by RT-PCR, western blot and ELISA. Results Dectin-1 mRNA and dectin-1 protein expression increased when HCECs were stimulated with A. fumigatus or curdlan, and CsA inhibited the dectin-1 expression both in mRNA and protein levels specifically. Dectin-1 and proinflammatory cytokine expression levels were higher when HCECs were pretreated with VDR inhibitor and CsA compared to pretreatment with CsA alone, while dectin-1 and proinflammatory cytokine levels were lower when HCECs were pretreated with 1,25(OH)2D3 and CsA compared to pretreatment with CsA alone. Conclusions These data provide evidence that CsA can inhibit the expression of dectin-1 and proinflammatory cytokines through dectin-1 when HCECs are stimulated by A. fumigatus or curdlan. The active form of vitamin D, 1,25(OH)2D3, and VDR signaling pathway regulate the inhibition of CsA. The inhibition is enhanced by 1,25(OH)2D3, and the VDR inhibitor suppresses the inhibition. PMID:27755569

  16. Human corneal epithelial subpopulations: oxygen dependent ex vivo expansion and transcriptional profiling.

    Science.gov (United States)

    Bath, Chris

    2013-06-01

    Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defective or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity. Since the first successful treatment of LSCD by transplantation of ex vivo expanded LESCs in 1997, many attempts have been carried out to optimize culture conditions to improve the outcome of surgery. To date, progress in this field of bioengineering is substantially hindered by both the lack of specific biomarkers of LESCs and the lack of a precise molecular characterization of in situ epithelial subpopulations. The aim of this dissertation was to optimize culture systems with regard to the environmental oxygen concentration for selective ex vivo expansion of LESCs and to analyse in situ subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum-free EpiLife medium, using a range of physiologically relevant oxygen concentrations (2%, 5%, 10%, 15% and 20%). Using immunocytochemistry and advanced fluorescence microscopy, cells were characterized regarding growth, cell cycle distribution, colony-forming efficiency (CFE), phenotypes and cytomorphometry. Limbal epithelial cells expanded in 2% O2 exhibited slow growth, low fraction of cells in S/G2 , high CFE, high expression of stem cell markers ABCG2 and p63α, and low fraction of differentiation marker CK3 resembling a LESC phenotype. The effect of hypoxia to maintain LESCs in culture was not dependent on the system used for propagation (Bath et al. 2013a). Laser capture microdissection was used to isolate cellular subpopulations in situ from the spatially defined

  17. Topical 100% Serum Eye Drops for Treating Corneal Epithelial Defect after Ocular Surgery

    Directory of Open Access Journals (Sweden)

    Kaevalin Lekhanont

    2013-01-01

    Full Text Available The purpose of this study was to investigate the efficacy and safety of topical 100% serum eye drops for corneal epithelial defect after ocular surgery. A total of 181 patients who received topical 100% serum therapy for the treatment of corneal epithelial defect following several different types of ocular surgery were recruited into this study. Each patient already failed conventional medical therapy before being prescribed 100% serum eye drops. Slit-lamp biomicroscopic examination with fluorescein staining was performed at baseline and all follow-up visits. The main outcome measures were the rate of complete healing of the corneal epithelial defect and incidence of adverse events. One hundred and seventy-eight eyes (98.34% received autologous serum eye drops, and 3 (1.66% received allogeneic serum eye drops. The overall success rate of treating persistent postoperative epithelial defect using 100% serum eye drops was 93.92% (95% CI 0.88–0.98. The median time to complete corneal epithelialization was 4 days (95% CI 4-5. Adverse reactions were observed in 3 patients (1.66%, including sticky sensation with minimal eye discomfort and asymptomatic trace corneal subepithelial infiltration. No serious complications were reported. In conclusion, 100% serum eye drops are effective, safe, and tolerable for treating postoperative corneal epithelial defect following ocular surgeries.

  18. Transport of L-carnitine in human corneal epithelial cells%左旋肉碱在人角膜缘上皮细胞的转运特性

    Institute of Scientific and Technical Information of China (English)

    李宝全; 毕建成; 安翠平; 李延峰; 许顺江

    2015-01-01

    Objective To investigate the characteristics of L-carnitine (LC)import into human corneal limbal epithelia (HCLE)cells,and to provide an experimental basis for further study of transport mechanism of LC in human ocular epithelium.Methods The transport of [3 H ]-L-carnitine was determined using the radio uptake assay and the apparent kinetic parameters of carnitine uptake by HCLE were estimated by nonlinear regression curve fitting according to the Michaelis-Menten equation.Results The uptake of LC into HCLE cells was saturable and time-dependent,and it also required the presence of Na+ in the external medium.An Eadie-Hofstee plot showed two distinct components:a high-and a low-affinity carnitine transport system in HCLE cells.The unlabelled LC and acetyl-L-carnitine competitively inhibited the uptake of [3 H]-L-carnitine by HCLE cells.Conclusion L-carnitine is transported into HCLE cells from tears by an active carrier mediated transport system and exerts its biological function.%目的:检测左旋肉碱(L-carnitine,LC)在人角膜缘上皮(human corneal limbal epithelia,HCLE)细胞的转运特性,为进一步阐明 HCLE细胞对 LC的转运机制提供实验依据。方法采用放射摄入实验检测 HCLE细胞对[3 H]-L-carnitine的转运功能,并利用米氏方程(Michaelis-Menten equation)分析计算其动力学参数。结果HCLE细胞对 LC的转运过程具有饱和性和时间依赖性,并且反应体系中需要 Na+的存在。Eadie-Hofstee作图提示 HCLE细胞存在高亲和力和低亲合力2个肉碱转运系统。非标记 LC和乙酰化左旋肉碱可竞争性抑制[3 H]-L-carnitine的转运过程。结论 HCLE细胞可通过主动转运过程将泪液中的左旋肉碱摄入细胞内,发挥其生物学功能。

  19. Corneal epithelial bullae after short-term wear of small diameter scleral lenses.

    Science.gov (United States)

    Nixon, Alex D; Barr, Joseph T; VanNasdale, Dean A

    2017-04-01

    Complications of scleral lens wear are not well documented or understood. While multiple studies focus on oxygen transmission during scleral lens wear and associated corneal swelling, little is known about the effects of varying scleral lens fitting relationships, especially when there is corneal interaction. Scleral lenses, by convention, are designed to completely clear the corneal surface and rest on the conjunctival and scleral tissue. However, some designs maximize oxygen transmission by reducing the lens diameter, thickness, and recommended corneal clearance.While the modifications increase oxygen transmission in any scleral lens design, they also distribute the lens mass closer to the limbus and make visualization of corneal clearance, especially narrow in the limbal region, more difficult. The sequelae from mechanical interaction between scleral lenses and the ocular surface, in particular the cornea, remain uncertain. This case series will describe corneal epithelial bullae, molding, and epithelial erosions as unintended scleral lens complications. These corneal changes corresponded to areas of contact lens-corneal bearing confirmed utilizing a combined scanning laser ophthalmoscopy (SLO) and anterior segment OCT. This case series will discuss epithelial bullae detection, their etiology, and suggestions for application of this information into scleral lens fitting protocols. Copyright © 2016 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  20. Evaluation of Epithelial Integrity with Various Transepithelial Corneal Cross-Linking Protocols for Treatment of Keratoconus

    Directory of Open Access Journals (Sweden)

    Suphi Taneri

    2014-01-01

    Full Text Available Purpose. Corneal collagen cross-linking (CXL has been demonstrated to stiffen cornea and halt progression of ectasia. The original protocol requires debridement of central corneal epithelium to facilitate diffusion of a riboflavin solution to stroma. Recently, transepithelial CXL has been proposed to reduce risk of complications associated with epithelial removal. Aim of the study is to evaluate the impact of various transepithelial riboflavin delivery protocols on corneal epithelium in regard to pain and epithelial integrity in the early postoperative period. Methods. One hundred and sixty six eyes of 104 subjects affected by progressive keratoconus underwent transepithelial CXL using 6 different riboflavin application protocols. Postoperatively, epithelial integrity was evaluated at slit lamp and patients were queried regarding their ocular pain level. Results. One eye had a corneal infection associated with an epithelial defect. No other adverse event including endothelial decompensation or endothelial damage was observed, except for epithelial damages. Incidence of epithelial defects varied from 0 to 63%. Incidence of reported pain varied from 0 to 83%. Conclusion. Different transepithelial cross-linking protocols have varying impacts on epithelial integrity. At present, it seems impossible to have sufficient riboflavin penetration without any epithelial disruption. A compromise between efficacy and epithelial integrity has to be found.

  1. Study of Topical Human Umbilical Cord Blood Serum in the Treatment of Alkaline Corneal Epithelial Wounds in Rabbit Model

    Directory of Open Access Journals (Sweden)

    B Sharifi

    2011-04-01

    Full Text Available Introduction & Objective: One of the important functions of the cornea is to maintain normal vision by refracting light onto the lens and retina. This property is dependent in part on the ability of the corneal epithelium to undergo continuous renewal. Ocular surface failure which follows a variety of endogenous and exogenous precipitating factors, the most common being: chemical trauma, infection, alkaline burn, inflammation and hereditary conditions, lid or lash abnormalities, tear deficiency or reduced sensation. The core principal underpinning management strategy for ocular surface failure is establishing or promoting new growth of healthy conjunctiva and corneal epithelium. This process is mediated by many proteins that are inducers of corneal cell migration, proliferation, and differentiation. The current study was performed to investigate the efficacy of umbilical cord serum on alkaline corneal epithelial wound healing in the rabbit model. Materials & Methods: In this study conducted at Yasuj University of Medical Sciences in 2010, thirty two rabbits were randomly assigned into two equal groups. Central corneal alkali wound was formed in one eye of the rabbits by applying a 6-mm round filter paper, soaked in 1 N NaOH, for 60 seconds. Group one of animals received umbilical cord blood serum and group two received Sno*Tear in the eyes. The treatment was dosed 4 times a day with the eye drops, and epithelial wound closure was recorded using slit lamp. The data were analyzed to determine the rate of wound closure. Results: The mean wound radius closure rate was 0.77 mm/day (SD=0.013 for umbilical cord blood serum-treated eyes, 0.73 mm/day (SD=0.018 for artificial tear-treated eyes. Conclusion: This study shows that alkali-injured corneal epithelial wound heal faster when treated with umbilical cord blood serum than with artificial tear in rabbit model.

  2. Evaluating alternative stem cell hypotheses for adultcorneal epithelial maintenance

    Institute of Scientific and Technical Information of China (English)

    John D West; Natalie J Dorà; Natalie J Dorà,

    2015-01-01

    In this review we evaluate evidence for three differenthypotheses that explain how the corneal epitheliumis maintained. The limbal epithelial stem cell (LESC)hypothesis is most widely accepted. This proposes thatstem cells in the basal layer of the limbal epithelium,at the periphery of the cornea, maintain themselvesand also produce transient (or transit) amplifying cells(TACs). TACs then move centripetally to the centre ofthe cornea in the basal layer of the corneal epitheliumand also replenish cells in the overlying suprabasallayers. The LESCs maintain the corneal epitheliumduring normal homeostasis and become more active torepair significant wounds. Second, the corneal epithelialstem cell (CESC) hypothesis postulates that, duringnormal homeostasis, stem cells distributed throughoutthe basal corneal epithelium, maintain the tissue.According to this hypothesis, LESCs are present in thelimbus but are only active during wound healing. We alsoconsider a third possibility, that the corneal epithelium ismaintained during normal homeostasis by proliferationof basal corneal epithelial cells without any input fromstem cells. After reviewing the published evidence,we conclude that the LESC and CESC hypotheses areconsistent with more of the evidence than the thirdhypothesis, so we do not consider this further. The LESCand CESC hypotheses each have difficulty accountingfor one main type of evidence so we evaluate the twokey lines of evidence that discriminate between them.Finally, we discuss how lineage-tracing experimentshave begun to resolve the debate in favour of theLESC hypothesis. Nevertheless, it also seems likely thatsome basal corneal epithelial cells can act as long-termprogenitors if limbal stem cell function is compromised.Thus, this aspect of the CESC hypothesis may have alasting impact on our understanding of corneal epithelialmaintenance, even if it is eventually shown that stemcells are restricted to the limbus as proposed by the

  3. Normal corneal endothelial cell density in Nigerians

    Directory of Open Access Journals (Sweden)

    Ewete T

    2016-03-01

    Full Text Available Temitope Ewete,1 Efeoghene Uchenna Ani,2 Adegboyega Sunday Alabi1 1MeCure Eye Center, Lagos, 2Department of Ophthalmology, University of Port Harcourt, Port Harcourt, Nigeria Aim: The aim of the study was to describe the corneal endothelial cell density of adults at the MeCure Eye Center and to determine the relationship between age, sex, and corneal endothelial cell density. Methods: This study was a retrospective study looking at those records of individuals who had undergone specular microscopy or corneal endothelial cell count measurement at the MeCure Eye Center. Results: The endothelial cell characteristics of 359 healthy eyes of 201 volunteers were studied. The mean corneal endothelial cell density (MCD was 2,610.26±371.87 cells/mm2 (range, 1,484–3,571 cells/mm2. The MCD decreased from 2,860.70 cells/mm2 in the 20–30-year age group to 2,493.06 cells/mm2 in the >70-year age group, and there was a statistically significant relationship between age and MCD with a P-value of <0.001. There was no statistically significant correlation between sex and corneal endothelial cell density (P=0.45. Conclusion: This study shows that endothelial cell density in Nigerian eyes is less than that reported in the Japanese, American, and Chinese eyes, and is comparable to that seen in Indian and Malaysian eyes. Keywords: corneal, endothelial cell density, Nigerian

  4. Comparative study of the effects of recombinant human epidermal growth factor and basic fibroblast growth factor on corneal epithelial wound healing and neovascularization in vivo and in vitro.

    Science.gov (United States)

    Yan, Limeng; Wu, Wei; Wang, Zhichong; Li, Chaoyang; Lu, Xiaohe; Duan, Hucheng; Zhou, Jin; Wang, Xiaoran; Wan, Pengxia; Song, Yiyue; Tang, Jing; Han, Yu

    2013-01-01

    This study was undertaken to investigate the effects of recombinant human epidermal growth factor (rhEGF) and basic fibroblast growth factor (bFGF) on corneal wound healing and neovascularization (CNV). The positive effects of 10 ng/ml rhEGF and bFGF on the proliferation of corneal epithelial cells (SD-HCEC1s), rabbit keratocyte cells (RKCs) and human umbilical vein endothelial cells (HUVECs) as well as the effects on the migration capacity on HUVECs were observed. An animal central corneal wound and CNV model was established in rabbits. One eye of each group was chosen randomly for topical administration of rhEGF, bFGF or normal saline, and variability in the area of corneal epithelial wound healing and CNV was observed. The optimal concentration of rhEGF and bFGF for the proliferation of corneal epithelial cells was 10 ng/ml. The promotive effect of 10 ng/ml rhEGF on the proliferation of RKCs and HUVECs was less than that of 10 ng/ml bFGF. In the animal experiment, the healing rate of the corneal epithelium in the rhEGF group was better than in the other groups on day 1. On day 3, the healing rates of the 3 groups were nearly equal. The CNV area in the rhEGF group was less than that of the bFGF group. rhEGF and bFGF both had promotive effects on corneal epithelial wound healing, but rhEGF had a weaker promotive effect on CNV than bFGF. With long-term application of growth factor drugs, rhEGF is suggested for lessening the growth of CNV. Copyright © 2012 S. Karger AG, Basel.

  5. Corneal reconstruction by stem cells and bioengineering

    Directory of Open Access Journals (Sweden)

    Arjamaa O

    2012-09-01

    Full Text Available Olli ArjamaaDepartment of Biology, University of Turku, Turku, FinlandAbstract: Almost 300 million people are visually impaired worldwide due to various eye diseases such as cataracts, glaucoma, age-related macular degeneration, diabetic retinopathy, and corneal diseases. Notably, ten million people are blind because of severe ocular surface diseases and the majority of cases occur in developing countries. Blinding ocular surface diseases have, however, become treatable by grafting of surface layers, or by full-thickness transplantation of the cornea. As the demand for human corneal tissue for surface reconstruction and transplantation far exceeds the supply, methods are being developed to supplement tissue donation. Xenotransplantation of the cornea or cells from genetically modified pigs may become one of the solutions. Transplantation of limbal stem cells within tissue biopsies, to restore the transparency of the cornea is another remarkable method, which has shown its potential in several clinical studies. The combination of stem cell technology and engineering of biocompatible tissue equivalent, still at preclinical stage, has shown us how synthetic corneal tissue is able to guide cultured corneal stromal stem cells of human origin, to become native-like stroma, the most important layer of the cornea. These findings give hope for a large-quantity production of biomaterial for corneal reconstruction. As such, clinical ophthalmologists should become more familiar with the methods of laboratory science.Keywords: eye, grafting, keratoplasty, xenotransplantation, cell reservoir, biocompatible tissue equivalent

  6. F{sub 2} excimer laser (157 nm) radiation modification and surface ablation of PHEMA hydrogels and the effects on bioactivity: Surface attachment and proliferation of human corneal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Zainuddin, E-mail: z.zainuddin@uq.edu.a [Queensland Eye Institute, 41 Annerley Road, South Brisbane, Queensland 4101 (Australia); University of Queensland, School of Medicine, Herston, Queensland 4006 (Australia); University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072 (Australia); Chirila, Traian V. [Queensland Eye Institute, 41 Annerley Road, South Brisbane, Queensland 4101 (Australia); University of Queensland, School of Medicine, Herston, Queensland 4006 (Australia); Queensland University of Technology, School of Physical and Chemical Sciences, Brisbane, Queensland 4001 (Australia); University of Queensland, Australian Institute for Bioengineering and Nanotechnology, St. Lucia, Queensland 4072 (Australia); Barnard, Zeke [Queensland Eye Institute, 41 Annerley Road, South Brisbane, Queensland 4101 (Australia); Watson, Gregory S. [James Cook University, School of Pharmacy and Molecular Sciences, Townsville, Queensland 4811 (Australia); Toh, Chiong; Blakey, Idriss [University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072 (Australia); University of Queensland, Australian Institute for Bioengineering and Nanotechnology, St. Lucia, Queensland 4072 (Australia); Whittaker, Andrew K. [Queensland Eye Institute, 41 Annerley Road, South Brisbane, Queensland 4101 (Australia); University of Queensland, Centre for Advanced Imaging, St. Lucia, Queensland 4072 (Australia); University of Queensland, Australian Institute for Bioengineering and Nanotechnology, St. Lucia, Queensland 4072 (Australia); Hill, David J.T. [The University of Queensland, School of Chemistry and Molecular Biosciences, St. Lucia, Queensland 4072 (Australia)

    2011-02-15

    Physical and chemical changes at the surface of poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels modified by ablation with an F{sub 2} excimer laser were investigated experimentally. An important observation was that only the outer exposed surface layers of the hydrogel were affected by the exposure to 157 nm radiation. The effect of the surface changes on the tendency of cells to adhere to the PHEMA was also investigated. A 0.5 cm{sup 2} area of the hydrogel surfaces was exposed to laser irradiation at 157 nm to fluences of 0.8 and 4 J cm{sup -2}. The changes in surface topography were analysed by light microscopy and atomic force microscopy, while the surface chemistry was characterized by attenuated total reflection infrared and X-ray photoelectron spectroscopies. Cell-interfacial interactions were examined based on the proliferation of human corneal limbal epithelial (HLE) cells cultured on the laser-modified hydrogels, and on the unexposed hydrogels and tissue culture plastic for comparison. It was observed that the surface topography of laser-exposed hydrogels showed rippled patterns with a surface roughness increasing at the higher exposure dose. The changes in surface chemistry were affected not only by an indirect effect of hydrogen and hydroxyl radicals, formed by water photolysis, on the PHEMA, but also by the direct action of laser radiation on PHEMA if the surface layers of the gel become depleted of water. The laser treatment led to a change in the surface characteristics, with a lower concentration of ester side-chains and the formation of new oxygenated species at the surface. The surface also became more hydrophobic. Most importantly, the surface chemistry and the newly created surface topographical features were able to improve the attachment, spreading and growth of HLE cells.

  7. Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy.

    Directory of Open Access Journals (Sweden)

    Haihui Liao

    Full Text Available BACKGROUND: Meesmann epithelial corneal dystrophy (MECD is an inherited eye disorder caused by dominant-negative mutations in either keratins K3 or K12, leading to mechanical fragility of the anterior corneal epithelium, the outermost covering of the eye. Typically, patients suffer from lifelong irritation of the eye and/or photophobia but rarely lose visual acuity; however, some individuals are severely affected, with corneal scarring requiring transplant surgery. At present no treatment exists which addresses the underlying pathology of corneal dystrophy. The aim of this study was to design and assess the efficacy and potency of an allele-specific siRNA approach as a future treatment for MECD. METHODS AND FINDINGS: We studied a family with a consistently severe phenotype where all affected persons were shown to carry heterozygous missense mutation Leu132Pro in the KRT12 gene. Using a cell-culture assay of keratin filament formation, mutation Leu132Pro was shown to be significantly more disruptive than the most common mutation, Arg135Thr, which is associated with typical, mild MECD. A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression with negligible effect on wild-type K12 or other closely related keratins. Cells transfected with wild-type K12-EGFP construct show a predominantly normal keratin filament formation with only 5% aggregate formation, while transfection with mutant K12-EGFP construct resulted in a significantly higher percentage of keratin aggregates (41.75%; p<0.001 with 95% confidence limits. The lead siRNA inhibitor significantly rescued the ability to form keratin filaments (74.75% of the cells contained normal keratin filaments; p<0.001 with 95% confidence limits. CONCLUSIONS: This study demonstrates that it is feasible to design highly potent si

  8. Umbilical cord blood serum therapy for the management of persistent corneal epithelial defects

    Institute of Scientific and Technical Information of China (English)

    Elif; Erdem; Meltem; Yagmur; Inan; Harbiyeli; Hande; Taylan-Sekeroglu; Reha; Ersoz

    2014-01-01

    AIM:Toevaluatetheroleofumbilical cord blood serum(CBS) therapy in cases with persistent corneal epithelial defects(PED).METHODS:Sixteen eyes of 14 patients with PED who were resistant to conventional treatment were treated with 20% umbilical cord serum eye drops. Patients were followed-up weekly until epithelization was complete.The collected data included the grade of corneal lesion(Grade I: epithelial defect +superficial vascularization,Grade II: epithelial defect +stromal edema, Grade III:corneal ulcer +stromal melting), the size of epithelial defect(pretreatment, 7th, 14 thand 21stdays of treatment),and follow-up time was evaluated retrospectively.RESULTS:The mean size of epithelial defect on two perpendicular axes was 5.2×4.6-mm2(range: 2.5-8 mm×2.2-9 mm2). Mean duration of treatment was 8.3 ±5wk.CBS therapy was effective in 12 eyes(75%) and ineffective in 4 eyes(25%). The epithelial defects in 4ineffective eyes were healed with amniotic membrane transplantation and tarsorrhaphy. The rate of complete healing was 12.5% by 7d, 25% by 14 d, and 75% by 21 d.The healing time was prolonged in Grade III eyes in comparison to eyes in Grade I or Grade II.CONCLUSION:The results of the current study indicated the safety effectiveness of CBS drops in the management of PED. The grade of disease seems have a role on the healing time.

  9. Neutrophil interactions with keratocytes during corneal epithelial wound healing: a role for CD18 integrins.

    Science.gov (United States)

    The purpose of this study was to determine the role of keratocytes and leukocyte beta(2) (CD18) integrins in neutrophil (PMN) migration through the corneal stroma after epithelial scrape injury. Using C57BL/6 wild-type and CD18(-/-) mice, corneas were excised at 6 hours (wild-type) or 24 hours (CD18...

  10. Polysaccharide hydrogel combined with mesenchymal stem cells promotes the healing of corneal alkali burn in rats.

    Directory of Open Access Journals (Sweden)

    Yifeng Ke

    Full Text Available Corneal chemical burns are common ophthalmic injuries that may result in permanent visual impairment. Although significant advances have been achieved on the treatment of such cases, the structural and functional restoration of a chemical burn-injured cornea remains challenging. The applications of polysaccharide hydrogel and subconjunctival injection of mesenchymal stem cells (MSCs have been reported to promote the healing of corneal wounds. In this study, polysaccharide was extracted from Hardy Orchid and mesenchymal stem cells (MSCs were derived from Sprague-Dawley rats. Supplementation of the polysaccharide significantly enhanced the migration rate of primarily cultured rat corneal epithelial cells. We examined the therapeutic effects of polysaccharide in conjunction with MSCs application on the healing of corneal alkali burns in rats. Compared with either treatment alone, the combination strategy resulted in significantly better recovery of corneal epithelium and reduction in inflammation, neovascularization and opacity of healed cornea. Polysaccharide and MSCs acted additively to increase the expression of anti-inflammatory cytokine (TGF-β, antiangiogenic cytokine (TSP-1 and decrease those promoting inflammation (TNF-α, chemotaxis (MIP-1α and MCP-1 and angiogenesis (VEGF and MMP-2. This study provided evidence that Hardy Orchid derived polysaccharide and MSCs are safe and effective treatments for corneal alkali burns and that their benefits are additive when used in combination. We concluded that combination therapy with polysaccharide and MSCs is a promising clinical treatment for corneal alkali burns and may be applicable for other types of corneal disorder.

  11. Corneal epithelial toxicity of antiglaucoma formulations: in vitro study of repeated applications

    Directory of Open Access Journals (Sweden)

    Meloni M

    2012-09-01

    Full Text Available Marisa Meloni,1 Giampiero Cattaneo,2 Barbara De Servi11VitroScreen In Vitro Research Laboratories, 2Thea Farma, Milan, ItalyBackground: By using a biologically relevant and sensitive three-dimensional model of human corneal epithelium and multiple endpoint analysis, assessment of the potential for eye irritation and long-term compatibility of four registered ophthalmological preparations, ie, Timolabak®, Timoptol®, Nyogel®, and Timogel®, was performed. This approach enables classification of the potential for irritation, discriminating between mildly irritant and non-irritant ocular substances.Methods: The exposure protocol included two time periods, ie, 24 hours (acute application and 72 hours (repeated applications twice daily. This approach allows assessment of not only the acute reaction but also possible recovery, as well as mimicking the potential cumulative effects associated with long-term application. Using benzalkonium chloride (BAK 0.01% as a positive control, the following parameters were quantified: cellular viability by MTT test, histological analysis by hematoxylin and eosin staining, passive release of interleukin-1a by enzyme-linked immunosorbent assay, and OCLN gene expression by quantitative real-time polymerase chain reaction.Results: Cell viability was reduced to under the 50% cutoff value after acute exposure (24 hours to BAK 0.01%, and after repeated application (72 hours of Timoptol and Nyogel. Histological analysis after acute exposure showed signs of superficial damage with all formulations, and severe changes after repeated applications of Timoptol, BAK 0.01%, and Nyogel. Timolabak and Timogel did not significantly alter the morphology of the human corneal epithelial cells after the different exposure times. Interleukin-1α release was greater than that for the negative control (>20 pg/mL and the positive control (BAK 0.01%, Nyogel, and Timoptol treatments and not different after treatment with Timolabak and

  12. Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Many researchers have employed the cryopreserved amniotic membrane(CAM) and corneal epithelial cells in the treatment of a severely damaged burned cornea, with corneal epithelial cells cultured on an amniotic membrane (AM). The lyophilized amniotic membrane (LAM) has a higher graft take and a longer shelf life; it is easier to store and safer because of gamma irradiation. Two Teflon rings(Ahn's supporter) were made for culturing the cells on the LAM, and were then used to support the LAM. To reconstruct a corneal layer composed of corneal fibroblasts and epithelium, the corneal fibroblasts were first cultivated on the stromal side of LAM for five days, followed by epithelial cells culture on the epithelial side, by using the air-liquid interface culture. The reconstructed corneal layer composed of corneal fibroblasts and corneal epithelial cells has a much healthier basal layer of corneal epithelium than the reconstructed corneal epithelium, which was got by using only corneal epithelial cells, and resembles the epithelium of normal corneas, without the horny layer. Thus, the reconstruction of the corneal layer by using a LAM is considered to be a good in vitro model, not only for its application in toxicological test kits, but also for transplantation in patients with a severely damaged cornea.

  13. The effect of subconjunctival platelet-rich plasma on corneal epithelial wound healing.

    Science.gov (United States)

    Tanidir, Suzan Tayfun; Yuksel, Nursen; Altintas, Ozgul; Yildiz, Demir Kursat; Sener, Ender; Caglar, Yusuf

    2010-06-01

    Single-dose subconjunctival platelet-rich plasma (sPRP) injections with or without antibiotic treatment were investigated for their effect on corneal epithelial wound healing in a rabbit model. A total of 24 rabbits were used in this study. After collecting intracardiac blood samples from 16 rabbits, platelet-rich plasma was obtained by centrifugation. Animals were divided into 3 groups. A mechanical corneal epithelial defect of 7-mm diameter was created on the central cornea of the right eye of each animal. Group 1 (n = 8) received only sPRP, whereas group 2 (n = 8) received sPRP with topical antibiotic throughout the study. The third group (n = 8) served as the control group. Each animal was examined daily under biomicroscope for 10 days. Epithelial defect was measured in horizontal and vertical diameter with a 1/100 sensitive micrometer, and area of the defect was calculated. One animal was sacrificed in each group on the third and seventh day, and all remaining animals were sacrificed on the 10th day. Corneal histopathology was investigated for epithelial regeneration, presence of inflammation, and structural integrity of fibroblasts. All data were statistically analyzed for difference between the study groups. Group 1 had a significantly smaller size in horizontal and vertical defect diameters on each day as compared with the control group. Healing of the epithelial defect in group 1 was found to be significantly different than that in the control group (P fibroblast migration, quicker epithelial regeneration, and less inflammation in group 1 as compared with the other 2 groups. sPRP seems to improve corneal epithelial wound healing. However, antibiotic and sPRP combination may have a retarded healing effect as compared with platelet-rich plasma alone.

  14. Corneal cell adhesion to contact lens hydrogel materials enhanced via tear film protein deposition.

    Directory of Open Access Journals (Sweden)

    Claire M Elkins

    Full Text Available Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS, borate buffered saline (BBS, or Sensitive Eyes Plus Saline Solution (Sensitive Eyes, either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo.

  15. A novel in vivo corneal trans-epithelial electrical resistance measurement device.

    Science.gov (United States)

    Uematsu, Masafumi; Mohamed, Yasser Helmy; Onizuka, Naoko; Ueki, Ryotaro; Inoue, Daisuke; Fujikawa, Azusa; Kitaoka, Takashi

    2015-01-01

    To develop a device that is capable of easily measuring corneal transepithelial electrical resistance (TER) and changes in the corneal barrier function. We had previously developed an in vivo method for measuring corneal TER using intraocular electrode. This method can be used to precisely measure the decline of the corneal barrier function after instillation of benzalkonium chloride (BAC). In order to lessen the invasiveness of that procedure, we further refined the method for measuring the corneal TER by developing electrodes that could be placed on the cornea and in the conjunctival sac instead of inserting them into the anterior chamber. TER was then calculated by subtracting the electrical resistance, which lacked the corneal epithelial input, from the whole electrical resistance that was measured between the electrodes. Slit lamp examination and scanning electron microscopy (SEM) were used to determine safety of the new device. Corneal TER changes after exposure to 0.02% BAC were determined using the new device as well as SEM and transmission electron microscopy (TEM). Slit lamp examination before and after exposure of rabbits' corneas to the sensor confirmed safety of the device. SEM examination revealed no difference of the corneal epithelium which exposed to the new device with normal corneas. SEM and TEM pictures revealed damaged microvilli and tight junctions after instillation of 0.02% BAC. TER change after treatment with 0.02%BAC was similar to those determined by the established anterior chamber method. We succeeded to develop a less invasive device for corneal TER measurement in vivo in animals. This new device may be applicable in the future for clinical use in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Differentiation of embryonic stem cells into corneal epithelium

    Institute of Scientific and Technical Information of China (English)

    WANG Zhichong; LIU Jingbo; GE Jian; HUANG Bing; GAO Qianying; LIU Bingqian; WANG Linghua; YU Ling; FAN Zhigang; LU Xiaoming

    2005-01-01

    Our project was to determine whether embryonic stem (ES) cells could be induced to differentiate into corneal epithelia by superficial corneoscleral limbal stroma. To achieve this goal, ES-GFP cell line D3 was pre-induced by retinoic acid (RA). The pre-induced cells were seeded on deepithelialized superficial corneoscleral slices (SCSS) to form a monolayer, and divided into three groups. Group 1 was cultured and passaged in vitro for direct detection. Group 2 was exposed to air-liquid interfaces for 10 days and implanted into the subcutaneous layer of nude mice for 2 weeks for further induction in vivo. Group 3 was cultured in vitro without any inducing factors for control. There were no teratomas found in nude mice which were implanted with differentiated ES cells after two weeks. The differentiated cells showed an appearance of epithelia both in vitro and in vivo. Expression of CK3, P63 and PCNA was detected by immunohistochemical staining in the differentiated cells in group 1 and 2. Microvillis and zonula occludens were observed on the surface of the differentiated cells under an electron microscope. In the control group, ES cells differentiated freely without any inducing factors. Most cells were shed and formed a neuronal dendrite-like structure, and a minority of cells appeared polymorphic. These results demonstrate that ES cells can differentiate into corneal epithelia on the surface of SCSS under the controlled condition. Differentiated ES cells could be used as epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering in the future.

  17. DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

    Science.gov (United States)

    Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

    2014-04-01

    DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

  18. Piroxicam与NS-398对角膜新生血管抑制作用及对兔角膜上皮毒性作用的比较%NS-398 inhibits corneal neovascularization with lower cytotoxicity on corneal epithelial cell than Piroxicam in rabbits

    Institute of Scientific and Technical Information of China (English)

    李少华; 张黎; 肖青; 胡燕华

    2004-01-01

    目的:比较Piroxicam(环氧化酶-1选择抑制剂)和NS-398(环氧化酶-2选择性抑制剂)对角膜新生血管的抑制作用并比较两者对角膜上皮细胞的毒性作用.方法:将含20u g脂多糖的缓释小丸植入兔角膜基质以诱导角膜新生血管.使用不同浓度的Piroxicam和NS-398滴眼剂滴眼,植入10d后分析角膜新生血管的面积.采用MTT法分析Piroxicam和NS-398对角膜上皮细胞的毒性作用.结果:0.01 u mol/L NS-398和口0.01 u mol/L Piroxicam组兔角膜新生血管生长浓密,其它组因抑制剂的不同及浓度的不同角膜新生血管面积有不同程度的减低.在0.5,5及50μmol/L时,NS-398较Piroxicam对角膜上皮细胞的毒性低.结论:环氧化酶-1特异性抑制剂和环氧化酶-2特异性抑制剂对角膜新生血管形成均有抑制作用.相同浓度时,NS-398的抑制效果强于Piroxicam.在高浓度时NS-398较Piroxicam对角膜上皮细胞的毒性低.%AIM: To compare the inhibitive effect of Piroxicam (cyclooxygenase-1 selective inhibitor, COX-1 inhibitor) and NS-398 (cyclooxygenase-2 selective inhibitor, COX-2 inhibitor) on corneal neovascularization and their cytotoxity on corneal epithelial cells in vitro.METHODS: Slow-release pellets containing lipopolysac charide 20 μ g were prepared and implanted into the rabbit' s corneal stroma near the limbus to induce neovascularization. Eye drops containing NS-398 or Piroxicam at various concentrations were applied. Ten days after the implantation of pellet, the neovascularization was examined and cytotoxicity of the two materials on comeal epithelium cells was analyzed by MTT assay.RESULTS: In the 0.01 μ mol/L Piroxicam group, therewas dense neovascularization. In the other groups, neovascularization decreased to different degrees, depending on the drug and concentration applied. The cytotoxicity of NS-398 was lower than that of Piroxicam at 0.5, 5 and 50 μ mol/L and the difference had statistical significance.CONCLUSION: Both

  19. Mammary epithelial cell

    DEFF Research Database (Denmark)

    Kass, Laura; Erler, Janine Terra; Dembo, Micah

    2007-01-01

    a repertoire of transmembrane receptors, of which integrins are the best characterized. Integrins modulate cell fate by reciprocally transducing biochemical and biophysical cues between the cell and the extracellular matrix, facilitating processes such as embryonic branching morphogenesis and lactation...... in the mammary gland. During breast development and cancer progression, the extracellular matrix is dynamically altered such that its composition, turnover, processing and orientation change dramatically. These modifications influence mammary epithelial cell shape, and modulate growth factor and hormonal...

  20. Silk film topography directs collective epithelial cell migration.

    Directory of Open Access Journals (Sweden)

    Brian D Lawrence

    Full Text Available The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography's edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization.

  1. Coronaviruses in polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Bekker, C P; Voorhout, W F; Horzinek, M C; Van der Ende, A; Strous, G J; Rottier, P J

    1995-01-01

    Coronaviruses have a marked tropism for epithelial cells. In this paper the interactions of the porcine transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV-A59) with epithelial cells are compared. Porcine (LLC-PK1) and murine (mTAL) epithelial cells were grown on permeable supp

  2. Corneal mucus plaques.

    Science.gov (United States)

    Fraunfelder, F T; Wright, P; Tripathi, R C

    1977-02-01

    Corneal mucus plaques adhered to the anterior corneal surface in 17 of 67 advanced cases of keratoconjunctivitis sicca. The plaques were translucent to opaque and varied in size and shape, from multiple isolated islands to bizarre patterns involving more than half the corneal surface. Ultrastructurally, they consisted of mucus mixed with desquamated degenerating epithelial cells and proteinaceous and lipoidal material. The condition may be symptomatic but can be controlled and prevented in most cases by topical ocular application of 10% acetylcysteine.

  3. Subclinical keratoconus detection by pattern analysis of corneal and epithelial thickness maps with optical coherence tomography.

    Science.gov (United States)

    Li, Yan; Chamberlain, Winston; Tan, Ou; Brass, Robert; Weiss, Jack L; Huang, David

    2016-02-01

    To screen for subclinical keratoconus by analyzing corneal, epithelial, and stromal thickness map patterns with Fourier-domain optical coherence tomography (OCT). Four centers in the United States. Cross-sectional observational study. Eyes of normal subjects, subclinical keratoconus eyes, and the topographically normal eye of a unilateral keratoconus patient were studied. Corneas were scanned using a 26,000 Hz Fourier-domain OCT system (RTVue). Normal subjects were divided into training and evaluation groups. Corneal, epithelial, and stromal thickness maps and derived diagnostic indices, including pattern standard deviation (PSD) variables and pachymetric map-based keratoconus risk scores, were calculated from the OCT data. Area under the receiver operating characteristic curve (AUC) analysis was used to evaluate the diagnostic accuracy of the indices. The study comprised 150 eyes of 83 normal subjects, 50 subclinical keratoconus eyes of 32 patients, and 1 topographically normal eye of a unilateral keratoconus patient. Subclinical keratoconus was characterized by inferotemporal thinning of the cornea, epithelium, and stroma. The PSD values for corneal (P keratoconus risk score (AUC = 0.735). High-resolution Fourier-domain OCT could map corneal, epithelial, and stromal thicknesses. Corneal and sublayer thickness changes in subclinical keratoconus could be detected with high accuracy using PSD variables. These new diagnostic variables might be useful in the detection of early keratoconus. Oregon Health and Science University (OHSU) and Drs. Li, Tan, and Huang have a significant financial interest in Optovue, Inc. These potential conflicts have been reviewed and managed by OHSU. Dr. Brass receives research grants from Optovue, Inc. Drs. Chamberlain and Weiss have no financial or proprietary interest in any material or method mentioned. Copyright © 2016 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  4. 低氧条件下人角膜上皮对棘阿米巴易感性的观察%Study on the susceptibility of human corneal epithelial cells to Acanthamoeba in a hypoxia condition

    Institute of Scientific and Technical Information of China (English)

    庞鵾鹏; 潘虹

    2016-01-01

    Objective To investigate the regulation mechanism of Acanthamoeba-induced immune responses of human corneal epithelial cells in a hypoxia condition.Methods Telomerase-immortalized human epithelial cells (THCEs) challenged with Acanthamoeba (1 × 106/ml) were incubated under normoxic (21% O2) (control group) or hypoxic (1% O2) (experiment group) conditions respectively.The mRNA of toll-like receptor (TLR4),myeloid differentiation factor 88 (MyD88),nuclear factor kappa-B (NF-κB),tumor necrosis factor-o (TNF-α),interleukin-8 (IL-8),interferon-β (IFN-β),and the protein level of TLR4,inhibitor of nuclear factor kappa-Bαt (IκBαt),phosphorylated extracellular-signal related kinase 1/2 (p-ERK1/2) and phosphorylated IκBα (p-IκBo) were detected.Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines (TNF-α,IL-8 and IFN-β).Results The mRNA levels of Acanthamoeba-induced TLR4,MyD88,NF-κB,TNF-α,IFN-β and IL-8 in THCEs under hypoxia was down-regulated by 47%,41%,45%,53%,36% and 50% respectively,compared to control group.And the protein levels of p-IκBα and p-ERK1/2 were significantly down-regulated by 56% and 55%,respectively,while the protein expression of IκBα was increased.The secretions of TNF-α,IL-8 and IFN-β under hypoxia were reduced by 46%,28% and 35%,respectively.Conclusion Hypoxia might attenuated the inflammatory response of the human corneal epithelial cells against Acanthamoeba infection by suppressing TLR4 signaling.%目的 探讨低氧条件下人角膜上皮对棘阿米巴易感性的调控机制.方法 培养永生化人角膜上皮细胞(THCEs),随机分为低氧(1%O2)棘阿米巴刺激组和常氧(21%O2)棘阿米巴刺激组(1×106/ml).收集刺激细胞检测Toll样受体4(TLR4)及其通路分子髓样分化因子88(MyD88)、核因子κB(NF-κB),以及肿瘤坏死因子α(TNF-α)、白细胞介素8(IL-8)和干扰素β(IFN-β) mRNA

  5. Treatment of Recurrent Corneal Epithelial Defect by Autologous Serum Eye Drop

    Directory of Open Access Journals (Sweden)

    Hossein Mohammad Rabei

    2015-02-01

    Full Text Available Background: The aim of the present study was to evaluate the efficacy of autologous serum eye drop in treatment of recurrent corneal epithelial defect.Materials and Methods: Fourteen patients with recurrent corneal epithelial defect were studied. Autologous serum was prepared from the patients and diluted in 20% normal saline. The patients were instructed to use the autologous serum every six hours. Patients were followed for a mean period of 18 months.Results: Four males (28.6% and 10 females (71.4% entered the study. Four patients stopped the treatment after three months with complete satisfaction from treatment. Patients reported a reduction in frequency and severity of attacks 4.6±2 weeks after the start of treatment. The mean number of attacks before the procedure was 7.6±0.9 per year which was reduced to 2.2±0.9 per year after treatment (p<0.001. The main side effects in patients were eye pruritus and redness which were well tolerated by patients.Conclusion: Autologous serum application seems to be a safe and effective method to treat recurrent corneal epithelial defect.

  6. Epidermal Growth Factor Receptor Transactivation by the Cannabinoid Receptor (CB1) and Transient Receptor Potential Vanilloid 1 (TRPV1) Induces Differential Responses in Corneal Epithelial Cells

    Science.gov (United States)

    2010-01-01

    inhibitors of proteolytic release of heparin bound EGF ( HB -EGF). CB1- induced Ca2þ transients were reduced during exposure to either the CB1 antagonist...blockage eliminated this response. Furthermore, EGFR transactivation was abolished by inhibitors of proteolytic release of heparin bound EGF ( HB -EGF...IL-8 or IL-6 Chemiluminescent Immunoassay ; R&D Systems, Minneapolis, MN). The cells were washed with basic medium and then exposed to CPZ, or AM251

  7. Comparison of corneal epithelial and stromal thickness distributions between eyes with keratoconus and healthy eyes with corneal astigmatism ≥ 2.0 D.

    Directory of Open Access Journals (Sweden)

    Wen Zhou

    Full Text Available PURPOSE: To identify corneal epithelial- and stromal-thickness distribution patterns in keratoconus using spectral-domain optical coherence tomography (SD-OCT. PATIENTS AND METHODS: We analyzed SD-OCT findings in 20 confirmed cases of keratoconus (group 1 and in 20 healthy subjects with corneal astigmatism ≥ 2 D (group 2. Epithelial and stromal thicknesses were measured at 11 strategic locations along the steepest and flattest meridians, previously located by corneal topography. Vertical mirrored symmetry superimposition was used in the statistical analysis. RESULTS: The mean maximum keratometry measurements in groups 1 and 2 were 47.9 ± 2.9 D (range, 41.8-52.8 and 45.6 ± 1.1 D (range, 42.3-47.5, respectively, with mean corneal cylinders of 3.3 ± 2.2 D (range, 0.5-9.5 and 3.6 ± 1.2 D (range, 2.0-6.4, respectively. The mean epithelial thickness along the steepest meridian in group 1 was the lowest (37.4 ± 4.4 µm at 1.2 mm inferotemporally and the highest (59.3 ± 4.4 µm at 1.4 mm supranasally from the corneal vertex. There was only a small deviation in thickness along the steepest meridian in group 2, as well as along the flattest meridians in both groups. The stromal thickness distribution in the two groups was similar to the epithelial, while the stromal thickness was generally lower in group 1 than in group 2. CONCLUSIONS: SD-OCT provides details about the distribution of corneal epithelial and stromal thicknesses. The epithelium and stroma in keratoconic eyes were thinner inferotemporally and thicker supranasally compared with control eyes. The distribution pattern was more distinct in epithelium than in stroma. This finding may help improve the early diagnosis of keratoconus. TRIAL REGISTRATION: ClinicalTrials.gov NCT02023619.

  8. Epithelial cells as alternative human biomatrices for comet assay

    Directory of Open Access Journals (Sweden)

    Emilio eRojas

    2014-11-01

    Full Text Available The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes.Over a thirty year period, the comet assay in epithelial cells has been litlle employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  9. Isolation and transplantation of corneal endothelial cell-like cells derived from in-vitro-differentiated human embryonic stem cells.

    Science.gov (United States)

    Zhang, Kai; Pang, Kunpeng; Wu, Xinyi

    2014-06-15

    The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell-conditioned medium. Within 1 week, CEC-like cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the N-cadherin/vimentin dual-positive population enriches for CEC-like cells. The isolated CEC-like cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and seeded onto posterior acellular porcine corneal matrix lamellae to construct the CEC-like cell sheets. Pump function parameters of the CEC-like cell sheets approximated those of human donor corneas. Importantly, when the CEC-like cell sheets were transplanted into the eyes of rabbit CE dysfunction models, the corneal transparency was restored gradually. In conclusion, CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CEC biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future.

  10. ROS, MAPK/ERK and PKC play distinct roles in EGF-stimulated human corneal cell proliferation and migration.

    Science.gov (United States)

    Huo, Y-N; Chen, W; Zheng, X-X

    2015-11-08

    Cornea is at the outermost surface of eye globe, and it easily receives damage from ultraviolet light exposure, physiology wounding, and infections. It is essential to understand the mechanisms controlling human corneal epithelial (HCE) cell proliferation and wound healing. Epidermal growth factor (EGF) could stimulate cell proliferation and migration in various cell types. Therefore, we investigated the roles and mechanisms of EGF on HCE cell proliferation and migration. CCK-8 kit and wound healing experiment were used to investigate HCE cell proliferation and cell migration, respectively. ROS activity was quantified by DCFDA and flow cytometry. Western blot and Q-PCR were performed to examine protein and RNA levels. EGF could promote HCE cell proliferation and migration in both physiology status and UV irradiation conditions, which is used to mimic the disease condition in human corneal epithelial cells. Interestingly, the promotion effect of EGF on HCE cell proliferation is mainly mediated by activated ROS signaling under disease condition. However, the EGF function is mediated by ROS and MAPK/ERK pathway in EGF-treated corneal epithelial cells in physiology status, in which ROS and MAPK/ERK pathway have no mutual influence on the other signaling pathway in EGF-stimulated corneal epithelial cells. We also revealed that MAPK/ERK pathway instead of ROS mediates EGF-stimulated HCE cell migration. Interestingly, we found that PKC proteins were downregulated by EGF in HCE cells that is partially mediated by ROS signaling, while PKC pathway was not involved in EGF-stimulated corneal cell proliferation and migration. EGF promotes human corneal cell proliferation and migration both in physiology and disease conditions, and ROS, MAPK/ERK and PKC pathways play different roles in these processes.

  11. Collagen based film with well epithelial and stromal regeneration as corneal repair materials: Improving mechanical property by crosslinking with citric acid

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xuan; Liu, Yang; Li, Weichang; Long, Kai; Wang, Lin; Liu, Sa; Wang, Yingjun [School of Materials Science and Engineering, South China University of Technology, Guangzhou (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou (China); Ren, Li, E-mail: psliren@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou (China)

    2015-10-01

    Corneal disease can lead to vision loss. It has become the second greatest cause of blindness in the world, and keratoplasty is considered as an effective treatment method. This paper presents the crosslinked collagen (Col)–citric acid (CA) films developed by making use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results showed that the Col–CA films had necessary optical performance, water content. The collagenase resistance of CA crosslinked films was superior to that of EDC crosslinked films. And CA5 film (Col:CA:EDC:NHS = 60:3:10:10) had the best mechanical properties. Cell experiments showed that CA5 film was non-cytotoxic and human corneal epithelial cells could proliferate well on the films. Lamellar keratoplasty showed that the CA5 film could be sutured in the rabbit eyes and was epithelialized completely in about 10 days, and the transparency was restored quickly in 30 ± 5 days. No inflammation and corneal neovascularization were observed at 6 months. Corneal stroma had been repaired; stromal cells and neo-stroma could be seen in the area of operation from the hematoxylin–eosin stained histologic sections and anterior segment optical coherence tomography images. These results indicated that Col–CA films were highly promising biomaterials that could be used in corneal tissue engineering and a variety of other tissue engineering applications. - Highlights: • Adding different amounts of citric acid could change the properties of films. • The crosslinked films had better mechanical property than non-modified films. • Crosslinked collagen–citric acid films could tolerate suture during operation. • The films showed good ability of epithelial and stromal repair.

  12. A reproducible automated segmentation algorithm for corneal epithelium cell images from in vivo laser scanning confocal microscopy.

    Science.gov (United States)

    Bullet, Julien; Gaujoux, Thomas; Borderie, Vincent; Bloch, Isabelle; Laroche, Laurent

    2014-06-01

    To evaluate an automated process to find borders of corneal basal epithelial cells in pictures obtained from in vivo laser scanning confocal microscopy (Heidelberg Retina Tomograph III with Rostock corneal module). On a sample of 20 normal corneal epithelial pictures, images were segmented through an automated four-step segmentation algorithm. Steps of the algorithm included noise reduction through a fast Fourier transform (FFT) band-pass filter, image binarization with a mean value threshold, watershed segmentation algorithm on distance map to separate fused cells and Voronoi diagram segmentation algorithm (which gives a final mask of cell borders). Cells were then automatically counted using this border mask. On the original image either with contrast enhancement or noise reduction, cells were manually counted by a trained operator. The average cell density was 7722.5 cells/mm(2) as assessed by automated analysis and 7732.5 cells/mm(2) as assessed by manual analysis (p = 0.93). Correlation between automated and manual analysis was strong (r = 0.974 [0.934-0.990], p mean difference in density of 10 cells/mm(2) and a limits of agreement ranging from -971 to +991 cells/mm(2) . Visually, the algorithm correctly found almost all borders. This automated segmentation algorithm is worth for assessing corneal epithelial basal cell density and morphometry. This procedure is fully reproducible, with no operator-induced variability. © 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  13. Mechanism of Corneal Endothelial Cells Lesion during Phacoemulsification and Aspiration

    Institute of Scientific and Technical Information of China (English)

    Songtao Yuan; Lina Xie; Qinghuai Liu; Nanrong Yuan

    2003-01-01

    Purpose: To evaluate the proportions of corneal endothelial lesion caused by differentfactors during phacoemulsification and aspiration.Methods: Fourteen cats (twenty eight eyes) were divided into four groups. The processedfactors were ultrasonic power, lens extraction by phacoemulsification or not, and lensextraction using different levels of ultrasonic power. The density of central cornealendothelial cells was measured before and after operation.Results: There is no statistic difference between pre-operation density and post-operationdensity for releasing ultrasonic power only without lens extraction group. But for the lensextraction group, there is difference in density of central corneal endothelial cells andthe higher level of ultrasonic power, the more the central corneal endothelial cells densitydecreased through operation.Conclusion: The primary factor that causes corneal endothelial lesion duringphacoemulsification and aspiration procedure is debris of lens nucleus, and the otherfactors cause the lesion of corneal endothelium in normal operations just in very smalldegree.

  14. Multidrug resistance-associated protein (MRP1, 2, 4 and 5) expression in human corneal cell culture models and animal corneal tissue.

    Science.gov (United States)

    Verstraelen, Jessica; Reichl, Stephan

    2014-07-07

    Preclinical studies addressing the transcorneal absorption of ophthalmic drugs are mainly performed using ex vivo animal corneas and in vitro corneal cell culture models, leaving open the question of transferability to humans in an in vivo situation. While passive drug absorption through corneal tissue is well understood, little is known about the expression of transporter proteins and active drug transport in human and animal corneas as well as corneal cell culture models. Therefore, the aim of this study was to conduct an expression analysis of four multidrug resistance-associated proteins (MRP1, 2, 4 and 5) in various in vitro and ex vivo corneal models, leading to a better understanding of the comparability of different corneal models regarding drug absorption and transferability to humans. Two well-established in vitro human corneal models, the HCE-T epithelial model and the more organotypic Hemicornea construct, both of which are based on the SV40 immortalized human corneal epithelial cell line HCE-T, were analyzed, as were excised rabbit and porcine cornea. Specimens of abraded epithelia from human donor corneas were also tested. MRP mRNA expression was determined via reverse transcriptase polymerase chain reaction. Protein expression was examined using Western blot experiments and immunohistochemistry. The functional activity of the MRP efflux transporter was detected in transport assays using specific marker and inhibitor substances. The functional expression of all of the tested MRP transporters was detected in the HCE-T epithelial model. Hemicornea constructs displayed a similar expression pattern for MRP1, 4 and 5, whereas no MRP2 protein expression or activity was detected. However, excised animal corneas exhibited different expression profiles. In porcine cornea, no functional expression of MRP1, 2, or 5 was observed, and we failed to detect MRP4 expression in rabbit cornea. The results suggest that MRP1, 2, 4, and 5 are expressed in the human corneal

  15. Integrins and epithelial cell polarity.

    Science.gov (United States)

    Lee, Jessica L; Streuli, Charles H

    2014-08-01

    Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell-matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical-basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity.

  16. Adhesion and metabolic activity of human corneal cells on PCL based nanofiber matrices.

    Science.gov (United States)

    Stafiej, Piotr; Küng, Florian; Thieme, Daniel; Czugala, Marta; Kruse, Friedrich E; Schubert, Dirk W; Fuchsluger, Thomas A

    2017-02-01

    In this work, polycaprolactone (PCL) was used as a basic polymer for electrospinning of random and aligned nanofiber matrices. Our aim was to develop a biocompatible substrate for ophthalmological application to improve wound closure in defects of the cornea as replacement for human amniotic membrane. We investigated whether blending the hydrophobic PCL with poly (glycerol sebacate) (PGS) or chitosan (CHI) improves the biocompatibility of the matrices for cell expansion. Human corneal epithelial cells (HCEp) and human corneal keratocytes (HCK) were used for in vitro biocompatibility studies. After optimization of the electrospinning parameters for all blends, scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and water contact angle were used to characterize the different matrices. Fluorescence staining of the F-actin cytoskeleton of the cells was performed to analyze the adherence of the cells to the different matrices. Metabolic activity of the cells was measured by cell counting kit-8 (CCK-8) for 20days to compare the biocompatibility of the materials. Our results show the feasibility of producing uniform nanofiber matrices with and without orientation for the used blends. All materials support adherence and proliferation of human corneal cell lines with oriented growth on aligned matrices. Although hydrophobicity of the materials was lowered by blending PCL, no increase in biocompatibility or proliferation, as was expected, could be measured. All tested matrices supported the expansion of human corneal cells, confirming their potential as substrates for biomedical applications.

  17. Assessment of Corneal Epithelial Thickness in Asymmetric Keratoconic Eyes and Normal Eyes Using Fourier Domain Optical Coherence Tomography

    Directory of Open Access Journals (Sweden)

    S. Catalan

    2016-01-01

    Full Text Available Purpose. To compare the characteristics of asymmetric keratoconic eyes and normal eyes by Fourier domain optical coherence tomography (OCT corneal mapping. Methods. Retrospective corneal and epithelial thickness OCT data for 74 patients were compared in three groups of eyes: keratoconic (n=22 and normal fellow eyes (n=22 in patients with asymmetric keratoconus and normal eyes (n=104 in healthy subjects. Areas under the curve (AUC of receiver operator characteristic (ROC curves for each variable were compared across groups to indicate their discrimination capacity. Results. Three variables were found to differ significantly between fellow eyes and normal eyes (all p<0.05: minimum corneal thickness, thinnest corneal point, and central corneal thickness. These variables combined showed a high discrimination power to differentiate fellow eyes from normal eyes indicated by an AUC of 0.840 (95% CI: 0.762–0.918. Conclusions. Our findings indicate that topographically normal fellow eyes in patients with very asymmetric keratoconus differ from the eyes of healthy individuals in terms of their corneal epithelial and pachymetry maps. This type of information could be useful for an early diagnosis of keratoconus in topographically normal eyes.

  18. The influence of surface topography of a porous perfluoropolyether polymer on corneal epithelial tissue growth and adhesion.

    Science.gov (United States)

    Evans, Margaret D M; Chaouk, Hassan; Wilkie, John S; Dalton, Beatrice A; Taylor, Sarah; Xie, Ruo Zhong; Hughes, Timothy C; Johnson, Graham; McFarland, Gail A; Griesser, Hans H; Steele, John G; Meijs, Gordon F; Sweeney, Deborah F; McLean, Keith M

    2011-12-01

    Design principles for corneal implants are challenging and include permeability which inherently involves pore openings on the polymer surface. These topographical cues can be significant to a successful clinical outcome where a stratified epithelium is needed over the device surface, such as with a corneal onlay or corneal repair material. The impact of polymer surface topography on the growth and adhesion of corneal epithelial tissue was assessed using porous perfluoropolyether membranes with a range of surface topography. Surfaces were characterised by AFM and XPS, and the permeability and water content of membranes was measured. Biological testing of membranes involved a 21-day in vitro tissue assay to evaluate migration, stratification and adhesion of corneal epithelium. Similar parameters were monitored in vivo by surgically implanting membranes into feline corneas for up to 5 months. Data showed optimal growth and adhesion of epithelial tissue in vitro when polymer surface features were below a 150 nm RMS value. Normal processes of tissue growth and adhesion were disrupted when RMS values approached 300 nm. Data from the in vivo study confirmed these findings. Together, outcomes demonstrated the importance of surface topography in the design of implantable devices that depend on functional epithelial cover.

  19. Development of new therapeutic modalities for corneal endothelial disease focused on the proliferation of corneal endothelial cells using animal models.

    Science.gov (United States)

    Koizumi, Noriko; Okumura, Naoki; Kinoshita, Shigeru

    2012-02-01

    This review describes our recent attempts to develop new therapeutic modalities for corneal endothelial disease using animal models including non-human primate model in which the proliferative ability of corneal endothelial cells is severely limited, as is the case in humans. First, we describe our attempt to develop new surgical treatments using cultivated corneal endothelial cells for advanced corneal endothelial dysfunction. It includes two different approaches; a "corneal endothelial cell sheet transplantation" with cells grown on a type-I collagen carrier, and a "cell-injection therapy" combined with the application of Rho-kinase (ROCK) inhibitor. Recently, it was reported that the selective ROCK inhibitor, Y-27632, promotes cell adhesion and proliferation and inhibits the apoptosis of primate corneal endothelial cells in culture. When cultivated corneal endothelial cells were injected into the anterior chamber of animal eyes in the presence of ROCK inhibitor, endothelial cell adhesion was promoted and the cells achieved a high cell density and a morphology similar to corneal endothelial cells in vivo. We are also trying to develop a novel medical treatment for the early phase of corneal endothelial disease by the use of ROCK inhibitor eye drops. In rabbit and monkey experiments using partial endothelial dysfunction models, corneal endothelial wound healing was accelerated by the topical application of ROCK inhibitor to the ocular surface, and resulted in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. We are now trying to advance the clinical application of these new therapies for patients with corneal endothelial dysfunction.

  20. Corneal Cell Morphology in Keratoconus: A Confocal Microscopic Observation

    Science.gov (United States)

    Ghosh, Somnath; Mutalib, Haliza Abdul; Kaur, Sharanjeet; Ghoshal, Rituparna; Retnasabapathy, Shamala

    2017-01-01

    Purpose To evaluate corneal cell morphology in patients with keratoconus using an in vivo slit scanning confocal microscope. Methods A cross-sectional study was conducted to evaluate the corneal cell morphology of 47 keratoconus patients and 32 healthy eyes without any ocular disease. New keratoconus patients with different disease severities and without any other ocular co-morbidity were recruited from the ophthalmology department of a public hospital in Malaysia from June 2013 to May 2014. Corneal cell morphology was evaluated using an in vivo slit-scanning confocal microscope. Qualitative and quantitative data were analysed using a grading scale and the Nidek Advanced Visual Information System software, respectively. Results The corneal cell morphology of patients with keratoconus was significantly different from that of healthy eyes except in endothelial cell density (P = 0.072). In the keratoconus group, increased level of stromal haze, alterations such as the elongation of keratocyte nuclei and clustering of cells at the anterior stroma, and dark bands in the posterior stroma were observed with increased severity of the disease. The mean anterior and posterior stromal keratocyte densities and cell areas among the different stages of keratoconus were significantly different (P 0.05) among the three stages of keratoconus. Conclusion Confocal microscopy observation showed significant changes in corneal cell morphology in keratoconic cornea from normal healthy cornea. Analysis also showed significant changes in different severities of keratoconus. Understanding the corneal cell morphology changes in keratoconus may help in the long-term monitoring and management of keratoconus. PMID:28894403

  1. Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model

    Directory of Open Access Journals (Sweden)

    Tanaka Y

    2016-08-01

    Full Text Available Yohei Tanaka,1,2 Jun Nakayama2 1Department of Plastic Surgery, Clinica Tanaka Plastic, Reconstructive Surgery and Anti-aging Center, 2Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan Background and objective: Humans are increasingly exposed to near-infrared (NIR radiation from both natural (eg, solar and artificial (eg, electrical appliances sources. Although the biological effects of sun and ultraviolet (UV exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues.Materials and methods: DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C. The water-filter allowed 1,000–1,800 nm wavelengths and excluded 1,400–1,500 nm wavelengths.Results: A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm2 irradiation (P<0.05.Conclusion: We found that NIR irradiation induced the

  2. Diurnal variation of tight junction integrity associates inversely with matrix metalloproteinase expression in Xenopus laevis corneal epithelium: implications for circadian regulation of homeostatic surface cell desquamation.

    Directory of Open Access Journals (Sweden)

    Allan F Wiechmann

    Full Text Available The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium.Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2, membrane type 1-MMP (MT1-MMP and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime.MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface

  3. Effect of substrate composition and alignment on corneal cell phenotype.

    Science.gov (United States)

    Phu, Donna; Wray, Lindsay S; Warren, Robert V; Haskell, Richard C; Orwin, Elizabeth J

    2011-03-01

    Corneal blindness is a significant problem treated primarily by corneal transplants. Donor tissue supply is low, creating a growing need for an alternative. A tissue-engineered cornea made from patient-derived cells and biopolymer scaffold materials would be widely accessible to all patients and would alleviate the need for donor sources. Previous work in this lab led to a method for electrospinning type I collagen scaffolds for culturing corneal fibroblasts ex vivo that mimics the microenvironment in the native cornea. This electrospun scaffold is composed of small-diameter, aligned collagen fibers. In this study, we investigate the effect of scaffold nanostructure and composition on the phenotype of corneal stromal cells. Rabbit-derived corneal fibroblasts were cultured on aligned and unaligned collagen type I fibers ranging from 50 to 300 nm in diameter and assessed for expression of α-smooth muscle actin, a protein marker upregulated in hazy corneas. In addition, the optical properties of the cell-matrix constructs were assessed using optical coherence microscopy. Cells grown on collagen scaffolds had reduced myofibroblast phenotype expression compared to cells grown on tissue culture plates. Cells grown on aligned collagen type I fibers downregulated α-smooth muscle actin protein expression significantly more than unaligned collagen scaffolds, and also exhibited reduced overall light scattering by the tissue construct. These results suggest that aligned collagen type I fibrous scaffolds are viable platforms for engineering corneal replacement tissue.

  4. Ex vivo corneal epithelial wound healing following exposure to ophthalmic nonsteroidal anti-inflammatory drugs

    Directory of Open Access Journals (Sweden)

    Keping Xu

    2011-02-01

    Full Text Available Keping Xu1, Mark McDermott1, Linda Villanueva2, Rhett M Schiffman2, David A Hollander21The Kresge Eye Institute, Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI, USA; 2Allergan, Inc., Irvine, CA, USAPurpose: Ketorolac 0.45% is a new formulation of topical ketorolac in which preservative (benzalkonium chloride, BAK was removed and carboxymethylcellulose (CMC was added to improve tolerability and reduce dosing frequency. This study compared the effects of ketorolac 0.45% on corneal wound healing to prior ketorolac formulations (0.4% and 0.5%, bromfenac 0.09%, and nepafenac 0.1%.Methods: Two parallel-group comparisons were performed in series. A 5-mm central epithelial wound was made in fresh porcine corneas. After 24 hours in minimum essential medium (MEM, corneas were incubated for 10 minutes with study drugs, Triton X-100 1% (positive control, or MEM (negative control, followed by 24 hours in MEM. The remaining wound area was stained, photographed, and quantified (pixels. Study 1 compared ketorolac 0.45% to ketorolac 0.4% and ketorolac 0.5%. Study 2 compared ketorolac 0.45% to bromfenac 0.09% and nepafenac 0.1%.Results: The mean (±SD original wound area was 200,506 ± 4,363 pixels, which was reduced to 59,509 ± 4850 at 48 hours after exposure to Triton X-100 1%. In study 1, the mean remaining wound areas at 48 hours in pixels were 2969 ± 1633 with MEM, 586 ± 299 with ketorolac 0.45% (significantly reduced, P < 0.05 vs all other treatments, 10,228 ± 7541 with ketorolac 0.4%, and 50,674 ± 33,409 with ketorolac 0.5% (significantly enlarged, P < 0.05 vs MEM. In study 2, the mean remaining wound areas at 48 hours were 565 ± 1263 with MEM, 322 ± 229 with ketorolac 0.45% (significantly reduced, P < 0.01 vs bromfenac 0.09% and nepafenac 0.1%, 29,093 ± 14,295 with bromfenac 0.09% (significantly enlarged, P < 0.01 vs MEM and 47,322 ± 13,736 with nepafenac 0.1% (significantly enlarged, P < 0.01 vs MEM and vs

  5. Treatment of persistent corneal epithelial defect with overnight wear of a prosthetic device for the ocular surface.

    Science.gov (United States)

    Lim, Pauline; Ridges, Ryan; Jacobs, Deborah S; Rosenthal, Perry

    2013-12-01

    To report experience in the treatment of persistent corneal epithelial defect using overnight wear of a prosthetic device for the ocular surface. Retrospective interventional case series. A clinical database of patients who underwent prosthetic replacement of the ocular surface ecosystem (PROSE) treatment from March 2003 to August 2008 was searched to identify patients treated for persistent corneal epithelial defect. In early 2003, overnight wear of a PROSE device and addition of commercially available, nonpreserved, topical ophthalmic moxifloxacin to the saline in the device reservoir became standard practice at this center when treating persistent corneal epithelial defect. Medical records were abstracted to obtain underlying diagnoses, previous treatments, days to re-epithelialization, and complications for subsequent analysis. PROSE treatment incorporating overnight wear, with adjunctive use of moxifloxacin, was employed in 20 eyes of 19 patients for a total of 372 days. Re-epithelialization occurred in 17 of 20 eyes. Median duration of treatment incorporating overnight wear was 8.5 days (range = 2-76 days). Healing occurred in ≤7 days in 12 eyes, 8-14 days in 3 eyes, and >14 days in 2 eyes (range = 1-35 days). There were no cases of microbial keratitis. Overnight wear of a PROSE device is effective in promoting healing of persistent corneal epithelial defect. In comparison to an earlier series from this center, the rate of microbial keratitis as a complication of treatment has been reduced with the use of a nonpreserved topical fourth-generation fluoroquinolone in the device reservoir. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Combined eye gel containing sodium hyaluronate and xanthan gum for the treatment of the corneal epithelial defect after pterygium surgery

    Directory of Open Access Journals (Sweden)

    Kocatürk T

    2015-08-01

    Full Text Available Tolga Kocatürk,1 Ataman Gençgönül,2 Faruk Balica,1 Mehmet Özbağcivan,1 Harun Çakmak1 1Department of Ophthalmology, Faculty of Medicine, Adnan Menderes University, Aydin, 2Department of Ophthalmology, Mevki Military Hospital, Ankara, Turkey Aim: The aim of this study is to compare the effectiveness of the ophthalmic gels containing hyaluronate, xanthan gum, and netilmycine with fusidic acid in terms of recovery periods of corneal epithelium in the patients who underwent pterygium surgery.Methods: Patients who underwent pterygium surgery were separated into two groups. Forty patients in group 1 were given eye gel containing sodium hyaluronate, xanthan gum, and netilmycine, and 40 patients in group 2 were given one drop of eye gel containing fusidic acid. The patients in both groups were examined at the 12th, 24th, and 48th hours in the postoperative period by using slit-lamp technique. The subjective complaints of the patients such as pain and stinging, and the recovery periods of the corneal epithelial defect were evaluated comparatively by fluorescein staining.Results: The mean ages were 26±8 years (range: 18–35 and 24±6 years (range: 18–33 in groups 1 and 2, respectively. The closure period of the corneal epithelial defect was observed as 24±8 hours (range: 16–42 and 36±12 hours (range: 18–48 in groups 1 and 2, respectively. The number of the patients suffering from subjective complaints such as pain and stinging in the first 12 hours was six and 29 in groups 1 and 2, respectively. The difference was significant both clinically and statistically (P=0.001.Conclusion: Combined eye gel containing hyaluronate, xanthan gum, and netilmycine accelerates the recovery of corneal epithelial defect and reduces the complaints of the patients, when compared to the fusidic acid gel. Combined eye gel should be considered as an option for the treatment of the corneal epithelial defect related with pterygium surgery. Keywords: iatrogenic

  7. Corneal cellular proliferation and wound healing

    OpenAIRE

    Gan, Lisha

    2000-01-01

    Background. Cellular proliferation plays an important role in both physiological and pathological processes. Epithelial hyperplasia in the epithelium, excessive scar formation in retrocorneal membrane formation and neovascularization are examples of excessive proliferation of cornea cells. Lack of proliferative ability causes corneal degeneration. The degree of proliferative and metabolic activity will directly influence corneal transparency and very evidently refractive res...

  8. Anterior Segment OpticalCoherence Tomography: Assisted Topographic Corneal Epithelial Thickness Distribution Imaging of a Keratoconus Patient

    Directory of Open Access Journals (Sweden)

    A. John Kanellopoulos

    2013-04-01

    Full Text Available Purpose: To evaluate safety, efficacy and ease of measurement of epithelial thickness in a keratoconic patient based on anterior segment optical coherence tomography (AS-OCT. Methods: A 25-year-old male patient, previously diagnosed with keratoconus, with highly asymmetric manifestation among the two eyes, was subjected to AS-OCT corneal epithelial imaging. We investigated epithelial thickness and epithelial topographic thickness distribution. Results: Mean epithelial thickness was 51.97 ± 0.70 for the less affected right eye (OD, and 55.65 ± 1.22 for the more affected left eye (OS. Topographic epithelial thickness variability for the OD was 1.53 ± 0.21 μm, while for the OS it was 9.80 ± 0.41 μm. Conclusions: This case further supports our previous findings with high-frequency ultrasound measurements of the increase in overall epithelial thickness in keratoconic eyes in comparison with normal eyes. AS-OCT further offers ease of use and possibly higher predictability of measurement. This case report, based on AS-OCT imaging, verifies increased overall epithelial thickness in keratoconic eyes, as introduced by a previous study [Kanellopoulos et al.: Clin Ophthalmol 2012;6:789-800], based on high-frequency scanning ultrasound biomicroscopy imaging.

  9. Derivation of Corneal Keratocyte-Like Cells from Human Induced Pluripotent Stem Cells

    Science.gov (United States)

    Naylor, Richard W.; McGhee, Charles N. J.; Cowan, Chad A.; Davidson, Alan J.; Holm, Teresa M.; Sherwin, Trevor

    2016-01-01

    Corneal diseases such as keratoconus represent a relatively common disorder in the human population. However, treatment is restricted to corneal transplantation, which only occurs in the most advanced cases. Cell based therapies may offer an alternative approach given that the eye is amenable to such treatments and corneal diseases like keratoconus have been associated specifically with the death of corneal keratocytes. The ability to generate corneal keratocytes in vitro may enable a cell-based therapy to treat patients with keratoconus. Human induced pluripotent stem cells (hiPSCs) offer an abundant supply of cells from which any cell in the body can be derived. In the present study, hiPSCs were successfully differentiated into neural crest cells (NCCs), the embryonic precursor to keratocytes, and then cultured on cadaveric corneal tissue to promote keratocyte differentiation. The hiPSC-derived NCCs were found to migrate into the corneal stroma where they acquired a keratocyte-like morphology and an expression profile similar to corneal keratocytes in vivo. These results indicate that hiPSCs can be used to generate corneal keratocytes in vitro and lay the foundation for using these cells in cornea cell-based therapies. PMID:27792791

  10. Chitosan-gelatin biopolymers as carrier substrata for limbal epithelial stem cells.

    Science.gov (United States)

    de la Mata, Ana; Nieto-Miguel, Teresa; López-Paniagua, Marina; Galindo, Sara; Aguilar, María Rosa; García-Fernández, Luis; Gonzalo, Sandra; Vázquez, Blanca; Román, Julio San; Corrales, Rosa María; Calonge, Margarita

    2013-12-01

    The aim of this work was to evaluate semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human limbal epithelial cells (hLECs). To that end, human corneal epithelial cells (HCE) were cultured onto different CH-G membranes. None of the polymers were cytotoxic and cell proliferation was higher when CH was functionalized with G. Expression levels of corneal epithelial markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) were better maintained in HCE cells grown on CH-G 20:80 membranes than other proportions. Consequently, CH-G 20:80 was chosen for the subsequent expansion of hLECs. Cells derived from limbal explants were successfully expanded on CH-G 20:80 membranes using a culture medium lacking components of non-human animal origin. The expression levels found for corneal (K3 and K12) and limbal epithelial stem cells (K15) specific markers were similar to or higher than those found in limbal cells grown onto the control substratum. Our results demonstrate that CH-G 20:80 membranes are suitable for the expansion and maintenance of stem cells derived from the limbal niche. These results strongly support the use of polymers as alternative substrata for the transplantation of cultivated limbal cells onto the ocular surface.

  11. Caveolin-1 associated adenovirus entry into human corneal cells.

    Directory of Open Access Journals (Sweden)

    Mohammad A Yousuf

    Full Text Available The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC, caused by viruses within human adenovirus species D (HAdV-D, is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with

  12. Cartographic system for spatial distribution analysis of corneal endothelial cells.

    Science.gov (United States)

    Corkidi, G; Márquez, J; García-Ruiz, M; Díaz-Cintra, S; Graue, E

    1994-07-01

    A combined cartographic and morphometric endothelium analyser has been developed by integrating the HISTO 2000 histological imaging and analysis system with a prototype human corneal endothelium analyser. The complete system allows the elaboration and analysis of cartographies of corneal endothelial tissue, and hence the in vitro study of the spatial distribution of corneal endothelial cells, according to their regional morphometric characteristics (cell size and polygonality). The global cartographic reconstruction is obtained by sequential integration of the data analysed for each microscopic field. Subsequently, the location of each microscopically analysed field is referred to its real position on the histologic preparation by means of X-Y co-ordinates; both are provided by micrometric optoelectronic sensors installed on the optical microscope stage. Some cartographies of an excised human corneal keratoconus button in vitro are also presented. These cartographic images allow a macroscopic view of endothelial cells analysed microscopically. Parametric colour images show the spatial distribution of endothelial cells, according to their specific morphometric parameters, and exhibit the variability in size and cellular shape which depend on the analysed area.

  13. [The irido-corneo-endothelial syndrome. The loss of the control of corneal endothelial cell cycle. A review].

    Science.gov (United States)

    Robert, A M; Renard, G; Robert, L; Bourges, J-L

    2013-04-01

    The three major symptoms of the irido-corneo-endothelial syndrome are the alterations of the corneal endothelium and of the iris with a loss of the regulation of the cell cycle, and the progressive obstruction of the irido-corneal angle. This rare pathology attacks mainly young adult women. Most of the symptoms and complications originate from the excessive proliferation of the corneal endothelial cells accompanied by the evolution of their phenotype towards that of the epithelial cells. In normal conditions the corneal endothelial cells do not divide, they are blocked in the G1 stage of the cell cycle, mainly because of the action of the inhibitors of cyclin-dependent kinases. Still these cells retain a good capacity for proliferation, which can be induced by the down-regulation of the expression of the inhibitors of the cyclin-dependent kinases. This proliferative capacity declines with age and is also different according to the localization of the cells: it is more intense with those originating from the central area then in those from the peripheral area of the cornea. The age-related decline of the proliferative capacity is not due to the shortening of the telomers, but to the stress-induced accelerated senescence of the cells.

  14. Trans epithelial corneal collagen crosslinking for progressive keratoconus: 6 months follow up.

    Science.gov (United States)

    De Bernardo, Maddalena; Capasso, Luigi; Tortori, Antonia; Lanza, Michele; Caliendo, Luisa; Rosa, Nicola

    2014-12-01

    To evaluate keratoconus biomechanical changes after transepithelial corneal collagen cross linking (TE CXL) using riboflavin and ultraviolet A (UVA). Second University of Naples, Naples, Italy. Prospective non comparative case series study. Patients with progressive keratoconus were examined, before and during a 6 months follow up after TE CXL, with a Pentacam, an Ocular Response Analyzer and an IOLMaster. Best corrected visual acuity (BCVA), refraction, corneal thinnest point (CTP), keratometry readings at the keratoconus apex (Kmax), axial eye length (AL), corneal volume (CV) anterior chamber volume (ACV), anterior chamber depth (ACD), corneal hysteresis (CH) and corneal resistance factor (CRF) were evaluated. Thirty-six eyes of 36 patients with progressive keratoconus were analyzed. Six months after treatment there was a significant improvement in BCVA (pkeratoconus, without affecting in negative way the corneal elasticity. Copyright © 2014 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  15. Corneal endothelial cell density and morphology and central corneal thickness in Guangxi Maonan and Han adolescent students of China

    Institute of Scientific and Technical Information of China (English)

    Hao; Liang; Hui-Yi; Zuo; Jin-Mao; Chen; Jie; Cai; Yu-Zhua; Qin; Yu-Ping; Huang; Ying-Ying; Chen; Dong-Yong; Tang; Shao-Jian; Tan

    2015-01-01

    AIM: To investigate the corneal endothelial cell density and morphology and central corneal thickness in the Guangxi Maonan and Han adolescent students of China.METHODS: Noncontact specular microscope(Topcon SP3000 P, Tokyo, Japan) was performed in 133 adolescent students of Maonan nationality(M:F 54:79)and 105 adolescent students of Han nationality(M:F 50:55),5 to 20 y of age, who were randomly selected from 3schools in Huanjiang Maonan Autonomous County of Guangxi Zhuang Autonomous Region of China.Parameters studied included endothelial cell density,mean cell area, coefficient of variation in cell size,percentage hexagonality and central corneal thickness. RESULTS: Endothelial cell density, mean cell area,coefficient of variation in cell size, percentage hexagonality and central corneal thickness in the study population were(2969.50 ±253.93) cells/mm2,(339.23 ±29.44) μm2,(29.96 ±4.07) %,(64.58 ±9.41) % and(523.71 ±32.82) μm in Maonan and(2998.26 ±262.65) cells/mm2,(336.11±30.07) μm2,(29.89±5.03) %,(64.91±11.64) % and(524.39 ±33.15) μm in Han, respectively. No significant differences were observed in endothelial cell density,mean cell area, coefficient of variation in cell size,percentage hexagonality and central corneal thickness between Maonan and Han(P =0.615, 0.659, 0.528, 0.551,0.999). In Maonan and Han, we found age was negatively correlated with endothelial cell density and percentagehexagonality and positively correlated with mean cell area and coefficient of variation in cell size. Negative correlation was also found between central corneal thickness and age in Han, whereas no correlation was found in Maonan. CONCLUSION: There were no differences between Maonan and Han in corneal endothelial cell density and morphology and central corneal thickness. In these two nationalities, there were statistically significant decrease in endothelial cell density and percentage hexagonality with increasing age and statistically significant increase in

  16. Corneal Endothelial Cell Density and Morphology in Healthy Turkish Eyes

    Directory of Open Access Journals (Sweden)

    Ceyhun Arıcı

    2014-01-01

    Full Text Available Purpose. To describe the normative values of corneal endothelial cell density, morphology, and central corneal thickness in healthy Turkish eyes. Methods. Specular microscopy was performed in 252 eyes of 126 healthy volunteers (M : F, 42 : 84. Parameters studied included mean endothelial cell density (MCD, mean cell area (MCA, coefficient of variation (CV in cell size, percentage of hexagonal cells, and central corneal thickness (CCT. Results. The mean age of volunteers was 44.3±13.5 (range, 20 to 70 years. There was a statistically significant decrease in MCD (P<0.001; correlation, −0.388 and percentage of hexagonal cells, (P<0.001; correlation, −0.199 with age. There was also a statistically significant increase in MCA (P<0.001; correlation, 0.363 with increasing age. There was no statistically significant difference in MCD, MCA, CV in cell size, percentage of hexagonal cells, and CCT between genders and there was also no significant difference in these parameters between fellow eyes of subjects. Conclusions. Normotive data for the endothelium in the Turkish population are reported. Endothelial cell density in the Turkish eyes is less than that described in the Japanese, American, Chinese, and Filipino eyes and higher than that described in Indian, Thai, and Iranian eyes.

  17. Differential effect of rebamipide on transmembrane mucin biosynthesis in stratified ocular surface epithelial cells.

    Science.gov (United States)

    Uchino, Yuichi; Woodward, Ashley M; Argüeso, Pablo

    2016-12-01

    Mucins are a group of highly glycosylated glycoproteins responsible for the protection of wet-surfaced epithelia. Recent data indicate that transmembrane mucins differ in their contribution to the protective function of the ocular surface, with MUC16 being the most effective barrier on the apical surface glycocalyx. Here, we investigated the role of the mucoprotective drug rebamipide in the regulation of transmembrane mucin biosynthesis using stratified cultures of human corneal and conjunctival epithelial cells. We find that the addition of rebamipide to corneal, but not conjunctival, epithelial cells increased MUC16 protein biosynthesis. Rebamipide did not affect the levels of MUC1, 4 and 20 compared to control. In these experiments, rebamipide had no effect on the expression levels of Notch intracellular domains, suggesting that the rebamipide-induced increase in MUC16 biosynthesis in differentiated corneal cultures is not regulated by Notch signaling. Overall these findings indicate that rebamipide induces the differential upregulation of MUC16 in stratified cultures of human corneal epithelial cells, which may have implications to the proper restoration of barrier function in ocular surface disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Discovery of molecular markers to discriminate corneal endothelial cells in the human body

    NARCIS (Netherlands)

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji; Clevers, J.C.; van de Wetering, M.L.

    2015-01-01

    The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant

  19. Discovery of molecular markers to discriminate corneal endothelial cells in the human body

    NARCIS (Netherlands)

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji; Clevers, J.C.; van de Wetering, M.L.

    2015-01-01

    The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant nu

  20. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency

    Directory of Open Access Journals (Sweden)

    Tor Paaske Utheim

    2016-03-01

    Full Text Available The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC, which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD. Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells.

  1. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency.

    Science.gov (United States)

    Utheim, Tor Paaske; Utheim, Øygunn Aass; Khan, Qalb-E-Saleem; Sehic, Amer

    2016-03-01

    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells.

  2. A histological study of rabbit corneas after transepithelial corneal crosslinking using partial epithelial photoablation or ethanol treatment

    Directory of Open Access Journals (Sweden)

    Mehmet Cuneyt Ozmen

    2014-12-01

    Full Text Available AIM: To evaluate the histological changes after transepithelial corneal crosslinking (CXL using partial thickness excimer laser ablation or epithelial ethanol application in an experimental rabbit study.METHODS: Right eyes of twenty-four rabbits were studied. Four eyes received total epithelial debridement (group I. Four eyes received partial thickness epithelial ablation with excimer laser (group II. Twelve eyes were treated with different durations (30s and 60s and concentrations (18% to 48% of ethanol (group III. Riboflavin was applied for 30min intervals along with topical proparacaine drops with benzalkonium chloride, and 370 nm irradiation was performed for 30min, while riboflavin was instilled every 3min. Four eyes (group IV received 48% ethanol for 30s without riboflavin and irradiation. Eyes were collected after 24h and examined histologically.RESULTS: All eyes in group I showed keratocyte loss in the superficial 300 µ of corneal storma. In group II, 1-4 layers of epithelium were preserved and no keratocyte loss occurred. In group III, CXL after treatment with ethanol up to 24% concentration and up to 60s revealed no keratocyte loss. CXL after treatment with 48% and higher ethanol concentrations yielded keratocyte loss in the superficial 200 µ to 300 µ of cornea.CONCLUSION: Incomplete excimer laser ablation of the epithelium or treatment with ethanol up to 24% concentration and up to 60s duration yielded no stromal keratocyte loss. To get the same histological appearance seen in epithelial debridement group, partial thickness excimer laser epithelial ablation or ethanol application is not adequate for transepithelial CXL.

  3. Downregulation of PTEN at Corneal Wound Sites Accelerates Wound Healing through Increased Cell Migration

    Science.gov (United States)

    Cao, Lin; Graue-Hernandez, Enrique O.; Tran, Vu; Reid, Brian; Pu, Jin; Mannis, Mark J.

    2011-01-01

    Purpose. The PI3K/Akt pathway is required for cell polarization and migration, whereas the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) has inhibitory effects on the PI3K/Akt pathway. The authors therefore hypothesized that wounding would downregulate PTEN and that this downregulation would enhance wound healing. Methods. In human corneal epithelial (HCE) cell monolayer and rat cornea scratch wound models, the authors investigated PTEN and Akt expression using Western blot and immunofluorescence analyses. The effects of PTEN and PI3K inhibitors dipotassium bisperoxo (picolinato) oxovanadate (bpv(pic)) and LY294002 on cell migration and wound closure were investigated using time-lapse imaging. Finally, the authors investigated the effect of PTEN inhibition on wound healing in whole rat eyes. Results. In HCE cell monolayer and rat cornea, PTEN was downregulated at the wound edges within 30 minutes of wounding. The downregulation of PTEN was causal in a simultaneous increase in Akt activation, which was responsible for a significant increase in individual cell migration rate from 8.8 μm/h to 17.3 μm/h. An increased migration rate was maintained for 20 hours. PTEN inhibition significantly enhanced the wound healing rate in the HCE cell monolayer from 10 minutes onward after treatment and reduced the healing time in eye organ culture from 30 to 20 hours. Conclusions. Injury to the corneal epithelium downregulates the expression of PTEN at wound edges, allowing increased PI3K/Akt signaling, thereby contributing to a significant enhancement of cell migration and wound healing. These results suggest that PTEN inhibition may be an effective treatment for corneal injury. PMID:21212174

  4. Cell therapy of congenital corneal diseases with umbilical mesenchymal stem cells: lumican null mice.

    Directory of Open Access Journals (Sweden)

    Hongshan Liu

    Full Text Available BACKGROUND: Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation. METHODOLOGY/PRINCIPAL FINDINGS: Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice. CONCLUSIONS/SIGNIFICANCE: Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.

  5. Optimization of Human Corneal Endothelial Cells for Culture: The Removal of Corneal Stromal Fibroblast Contamination Using Magnetic Cell Separation

    Directory of Open Access Journals (Sweden)

    Gary S. L. Peh

    2012-01-01

    Full Text Available The culture of human corneal endothelial cells (CECs is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated “labeled” and “flow-through” cell fractions were collected separately, cultured, and morphologically assessed. Cells from the “flow-through” fraction displayed compact polygonal morphology and expressed Na+/K+ATPase indicative of corneal endothelial cells, whilst cells from the “labeled” fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs.

  6. Experiment Study of Effect of Perfiuorohexyloctane on Corneal Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan Ding; Chunfang Li; Lin Lu; Guanguang Feng; Huling Zheng

    2001-01-01

    Purpose: To investigate the effect of Perfluorohexyloctane (F6H8)on corneal endothelial celIs(CEC) of rabbit eyes. Methods: Fifteen New Zealand white rabbits were devided into two groups:experimental group(F6H8) and control group(BSS) . All rabbits underwent anterior chamber injection of 0. 15ml F6H8 or BSS. Slit-lamp biomicroscopy and corneal endothelium photography were performed pre-operatively and postoperatively. Histopathological examination and Transmission electron microscopy(TEM) were done after the rabbits were sacrificed. Results: All the corneas were clear. Since 4 weeks after operation, the endothelial cells were markedly irregular in size and shape and the number of endothelial cells was markedly decreased. Multilayered retrocorneal membranes (RCM)grew gradually 2 weeks after surgery. Vacuolar degeneration was seen in some endothelial cells. Nuclear degeneration and edema of plasma were seen in TEM. Conclusion: Corneal endothelial cell degenerated after contacting with F6H8 for 2 ~4weeks. As a silicone solvent, it should be removed completely after injection. We don't recommend it to be used as a new intraocular temponade. Eye Science 2001: 17:21 ~ 26.

  7. Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

    Science.gov (United States)

    Hertsenberg, Andrew J; Funderburgh, James L

    2016-01-01

    Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

  8. Role of Pattern Recognition Receptors in the Modulation of Antimicrobial Peptide Expression in the Corneal Epithelial Innate Response to F. solani

    Science.gov (United States)

    Kolar, Satya Sree; Baidouri, Hasna; McDermott, Alison M.

    2017-01-01

    Purpose Fusarium solani (F. solani) keratitis is a potentially sight-threatening fungal infection of the cornea. Antimicrobial peptides (AMPs), such as human β-defensins (hBDs) and cathelicidins, essential components of the immune system, likely have a protective role against F. solani keratitis. We examined the role of pattern recognition receptors (PRRs), Dectin-1, and TLR2 in F. solani–induced modulation of AMP expression in vitro. Methods Human corneal epithelial cells (HCECs) were exposed to heat-inactivated F. solani or pathogen-associated molecular patterns (PAMPs) of F. solani (Zymosan or Zymosan Depleted) for 6, 12, or 24 hours following which AMP mRNA and protein levels were determined. Involvement of TLR2 and Dectin-1 was confirmed by using siRNA knock-down (TLR2 and Dectin-1) or chemical inhibitor BAY 61-3606 (Dectin-1). The functional significance of AMP upregulation was tested using culture supernatant from F. solani or PAMP-treated HCECs against F. solani in the presence of hBD2 or LL37 neutralizing antibody. Results We confirm that HCECs express Dectin-1 and TLR2. HCECs demonstrated upregulation of AMPs hBD2 and cathelicidin LL37 following exposure to heat-inactivated F. solani or PAMPs. TLR2 and Dectin-1 knockdown and BAY 61-3606 treatment decreased AMP mRNA upregulation confirming PRR involvement. The culture supernatant from F. solani or PAMP-treated HCECs showed substantial killing of F. solani and hBD2 or LL37 neutralizing antibody significantly decreased this effect implicating involvement of these AMPs. Conclusions These findings demonstrate that Dectin-1 and TLR2 have an important role in regulating F. solani-induced AMP expression in corneal epithelial cells. PMID:28460048

  9. Feline corneal disease.

    Science.gov (United States)

    Moore, Phillip Anthony

    2005-05-01

    The cornea is naturally transparent. Anything that interferes with the cornea's stromal architecture, contributes to blood vessel migration, increases corneal pigmentation, or predisposes to corneal edema, disrupts the corneas transparency and indicates corneal disease. The color, location, and shape and pattern of a corneal lesion can help in determining the underlying cause for the disease. Corneal disease is typically divided into congenital or acquired disorders. Congenital disorders, such as corneal dermoids are rare in cats, whereas acquired corneal disease associated with nonulcerative or ulcerative keratitis is common. Primary ocular disease, such as tear film instability, adenexal disease (medial canthal entropion, lagophthalmus, eyelid agenesis), and herpes keratitis are associated with the majority of acquired corneal disease in cats. Proliferative/eosinophilic keratitis, acute bullous keratopathy, and Florida keratopathy are common feline nonulcerative disorders. Nonprogressive ulcerative disease in cats, such as chronic corneal epithelial defects and corneal sequestration are more common than progressive corneal ulcerations.

  10. Patterning bacterial communities on epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Dwidar

    Full Text Available Micropatterning of bacteria using aqueous two phase system (ATPS enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions.

  11. Corneal Regeneration by Deep Anterior Lamellar Keratoplasty (DALK Using Decellularized Corneal Matrix.

    Directory of Open Access Journals (Sweden)

    Yoshihide Hashimoto

    Full Text Available The purpose of this study is to demonstrate the feasibility of DALK using a decellularized corneal matrix obtained by HHP methodology. Porcine corneas were hydrostatically pressurized at 980 MPa at 10°C for 10 minutes to destroy the cells, followed by washing with EGM-2 medium to remove the cell debris. The HHP-treated corneas were stained with H-E to assess the efficacy of decellularization. The decellularized corneal matrix of 300 μm thickness and 6.0 mm diameter was transplanted onto a 6.0 mm diameter keratectomy wound. The time course of regeneration on the decellularized corneal matrix was evaluated by haze grading score, fluorescein staining, and immunohistochemistry. H-E staining revealed that no cell nuclei were observed in the decellularized corneal matrix. The decellularized corneal matrices were opaque immediately after transplantation, but became completely transparent after 4 months. Fluorescein staining revealed that initial migration of epithelial cells over the grafts was slow, taking 3 months to completely cover the implant. Histological sections revealed that the implanted decellularized corneal matrix was completely integrated with the receptive rabbit cornea, and keratocytes infiltrated into the decellularized corneal matrix 6 months after transplantation. No inflammatory cells such as macrophages, or neovascularization, were observed during the implantation period. The decellularized corneal matrix improved corneal transparency, and remodelled the graft after being transplanted, demonstrating that the matrix obtained by HHP was a useful graft for corneal tissue regeneration.

  12. Progress in corneal wound healing.

    Science.gov (United States)

    Ljubimov, Alexander V; Saghizadeh, Mehrnoosh

    2015-11-01

    Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β (TGF-β) system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal

  13. Progress in corneal wound healing

    Science.gov (United States)

    Ljubimov, Alexander V.; Saghizadeh, Mehrnoosh

    2015-01-01

    Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and

  14. Cell pattern in adult human corneal endothelium.

    Directory of Open Access Journals (Sweden)

    Carlos H Wörner

    Full Text Available A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.

  15. Airway epithelial cell responses to ozone injury

    Energy Technology Data Exchange (ETDEWEB)

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu [Univ. of Cincinnati Medical Center, OH (United States)] [and others

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

  16. Corneal endothelial cell density and morphology in Phramongkutklao Hospital

    Directory of Open Access Journals (Sweden)

    Narumon Sopapornamorn

    2008-03-01

    Full Text Available Narumon Sopapornamorn1, Manapon Lekskul1, Suthee Panichkul21Department of Ophthalmology, Phramongkutklao Hospital, Bangkok, Thailand; 2Department of Obstetrics and Gynecology, Phramongkutklao College of Medicine, Bangkok, ThailandObjective: To describe the corneal endothelial density and morphology in patients of Phramongkutklao Hospital and the relationship between endothelial cell parameters and other factors.Methods: Four hundred and four eyes of 202 volunteers were included. Noncontact specular microscopy was performed after taking a history and testing the visual acuity, intraocular pressure measurement, Schirmer’s test and routine eye examination by slit lamp microscope. The studied parameters included mean endothelial cell density (MCD, coefficient of variation (CV, and percentage of hexagonality.Results: The mean age of volunteers was 45.73 years; the range being 20 to 80 years old. Their MCD (SD, mean percentage of CV (SD and mean (SD percentage of hexagonality were 2623.49(325 cell/mm2, 39.43(8.23% and 51.50(10.99%, respectively. Statistically, MCD decreased significantly with age (p < 0.01. There was a significant difference in the percentage of CV between genders. There was no statistical significance between parameters and other factors.Conclusion: The normative data of the corneal endothelium of Thai eyes indicated that, statistically, MCD decreased significantly with age. Previous studies have reported no difference in MCD, percentage of CV, and percentage of hexagonality between gender. Nevertheless, significantly different percentages of CV between genders were presented in this study.Keywords: Corneal endothelial cell, parameters, age, gender, smoking, Thailand

  17. Aloe vera extract activity on human corneal cells.

    Science.gov (United States)

    Woźniak, Anna; Paduch, Roman

    2012-02-01

    Ocular diseases are currently an important problem in modern societies. Patients suffer from various ophthalmologic ailments namely, conjunctivitis, dry eye, dacryocystitis or degenerative diseases. Therefore, there is a need to introduce new treatment methods, including medicinal plants usage. Aloe vera [Aloe barbadensis Miller (Liliaceae)] possesses wound-healing properties and shows immunomodulatory, anti-inflammatory or antioxidant activities. NR uptake, MTT, DPPH• reduction, Griess reaction, ELISA and rhodamine-phalloidin staining were used to test toxicity, antiproliferative activity, reactive oxygen species (ROS) reduction, nitric oxide (NO) and cytokine level, and distribution of F-actin in cells, respectively. The present study analyzes the effect of Aloe vera extracts obtained with different solvents on in vitro culture of human 10.014 pRSV-T corneal cells. We found no toxicity of ethanol, ethyl acetate and heptane extracts of Aloe vera on human corneal cells. No ROS reducing activity by heptane extract and trace action by ethanol (only at high concentration 125 µg/ml) extract of Aloe vera was observed. Only ethyl acetate extract expressed distinct free radical scavenging effect. Plant extracts decreased NO production by human corneal cells as compared to untreated controls. The cytokine (IL-1β, IL-6, TNF-α and IL-10) production decreased after the addition of Aloe vera extracts to the culture media. Aloe vera contains multiple pharmacologically active substances which are capable of modulating cellular phenotypes and functions. Aloe vera ethanol and ethyl acetate extracts may be used in eye drops to treat inflammations and other ailments of external parts of the eye such as the cornea.

  18. Collection of corneal impression cytology directly on a sterile glass slide for the detection of viral antigen: An inexpensive and simple technique for the diagnosis of HSV epithelial keratitis – A pilot study

    Directory of Open Access Journals (Sweden)

    Bandlapally Sesha

    2001-09-01

    Full Text Available Abstract Background Herpes simplex keratitis (HSK is a sight threatening ocular infection and occurs worldwide. A prompt laboratory diagnosis is often very useful. Conventional virology techniques are often expensive and time consuming. We describe here a highly economical, simple, rapid and sensitive technique for the collection of impression cytology, for the laboratory diagnosis of HSK. Methods Fifteen patients with a clinical diagnosis of HSK (either dendritic or geographic ulcers and five patients with other corneal infections (Mycotic keratitis, n = 3, Bacterial keratitis, n = 2 were included in the study. Corneal impression cytology specimens were collected using a sterile glass slide with polished edges instead of a membrane, by pressing the surface of one end of the slide firmly, but gently on the corneal lesion. Additionally, corneal scrapings were collected following the impression cytology procedure. Impression cytology and corneal scrapings were stained by an immunoperoxidase or immunofluorescence assay for the detection of HSV-1 antigen using a polyclonal antibody to HSV-1. Corneal scrapings were processed for viral cultures by employing a shell vial assay. Results This simple technique allowed the collection of adequate corneal epithelial cells for the detection of HSV-1 antigen in a majority of the patients. HSV-1 antigen was detected in 12/15 (80% cases while virus was isolated from 5/15 (33.3% patients with HSK. All the patients with a clinical diagnosis of HSK (n = 15 were confirmed by virological investigations (viral antigen detection and/or viral cultures. HSV-1 antigen was detected in the impression cytology smears and corneal scrapings in 11/15 (73.3% and 12/15 (80% of the patients, respectively (P = 1.00. None of the patients in the control group were positive for viral antigen or virus isolation. Minimal background staining was seen in impression cytology smears, while there was some background staining in corneal

  19. Genomics of corneal wound healing: a review of the literature.

    Science.gov (United States)

    Maycock, Nick J R; Marshall, John

    2014-05-01

    Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.

  20. 兔角膜上皮细胞广泛丧失对角膜形态的影响%Effect of Epithelial Injury on Corneal Morphology

    Institute of Scientific and Technical Information of China (English)

    张阳; 聂庆珠; 盖春柳; 徐旭; 尹树国

    2001-01-01

    目的:观察兔角膜上皮广泛丧失对角膜形态的影响。方法:用大耳白兔20只,随机分为两组,左眼为实验眼,右眼作对照眼。将一组兔左眼角膜中央8 mm上皮刮除,另一组兔左眼角膜上皮广泛刮除,仅保留角膜缘部0.2 mm上皮。术后每日观察角膜上皮愈合情况,4周后测量角膜厚度、前房深度、角膜中央曲率。结果:广泛刮除角膜上皮眼上皮愈合时间明显延长,角膜厚度明显变薄,前房深度明显变深,角膜中央曲率明显变大。结论:兔角膜上皮广泛丧失可使角膜变薄,引起角膜曲率变大。%Objective: Our purpose was to observe the effect of epithelial scrape injury on corneal morphology. Methods: Twenty 4-week-old white rabbits were used. We scraped the corneal epithelia of the left eye of each rabbit (0.2 mm near the limbus of corneal were left in 10 eyes, in the remaining rabbits within 8 mm in the center). The right eyes were control group. We observed the healing of corneal protrusion with slit-lamp microscope, examined the corneal form with corneal topography, and measured the depth of anterior chamber and the corneal thickness with A-ultrasound. Results: The extensive epithelial scrape significantly increased the healing time. The corneal protrusion of experimental group and the depth of anterior chamber increased. The corneal thickness became thinner. Conclusion: The extensive epithelial injury can make cornea thinner, which results in the changes of corneal protrusion.

  1. Cultured corneas show dendritic spread and restrict herpes simplex virus infection that is not observed with cultured corneal cells

    Science.gov (United States)

    Thakkar, Neel; Jaishankar, Dinesh; Agelidis, Alex; Yadavalli, Tejabhiram; Mangano, Kyle; Patel, Shrey; Tekin, Sati Zeynep; Shukla, Deepak

    2017-01-01

    Herpes simplex virus-1 (HSV-1) causes life-long morbidities in humans. While fever blisters are more common, occasionally the cornea is infected resulting in vision loss. A very intriguing aspect of HSV-1 corneal infection is that the virus spread is normally restricted to only a small fraction of cells on the corneal surface that connect with each other in a dendritic fashion. Here, to develop a comprehensive understanding of the susceptibility of human corneal epithelial (HCE) cells to HSV-1 infection, we infected HCE cells at three different dosages of HSV-1 and measured the outcomes in terms of viral entry, gene and protein expression, viral replication and cytokine induction. In cultured cells, infectivity and cytokine induction were observed even at the minimum viral dosage tested, while a more pronounced dose-restricted infectivity was seen in ex vivo cultures of porcine corneas. Use of fluorescent HSV-1 virions demonstrated a pattern of viral spread ex vivo that mimics clinical findings. We conclude that HCE cell cultures are highly susceptible to infection whereas the cultured corneas demonstrate a higher ability to restrict the infection even in the absence of systemic immune system. The restriction is helped in part by local interferon response and the unique cellular architecture of the cornea. PMID:28198435

  2. Dry Eye and Corneal Langerhans Cells in Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Miklós D. Resch

    2015-01-01

    Full Text Available Purpose. Investigation of dry eye and corneal Langerhans cells (LCs in systemic lupus erythematosus (SLE. Methods. Prospective consecutive case series of 27 SLE patients and 27 control subjects. Dry eye was evaluated by lid-parallel conjunctival folds (LIPCOF, Schirmer test, tear break-up time (TBUT, and ocular surface disease index (OSDI questionnaire. In vivo investigation of corneal LCs density and morphology (LCM was performed with confocal corneal microscopy (Heidelberg Retina Tomograph with Rostock Cornea Module. Results. Tear production and stability were pathological in SLE subjects compared to control (Schirmer: 8.45 ± 9.82 mm/5 min versus 11.67 ± 3.21 mm/5 min; TBUT: 6.86 ± 3.53 s versus 11.09 ± 3.37 s. OSDI was significantly greater in SLE patients (25.95 ± 17.92 than in controls (11.06 ± 7.18. Central LC density was greater in SLE patients (43.08 ± 48.67 cell/mm2 than in controls (20.57 ± 21.04 cell/mm2. There was no difference in the peripheral LC density (124.78 ± 165.39 versus 78.00 ± 39.51 cell/mm2. LCM was higher in SLE patients in the centre (1.43 ± 0.79 and in the periphery (2.89 ± 0.42 compared to controls (centre: 1.00 ± 0.69, periphery: 2.35 ± 0.54. Conclusions. Significant changes in dry eye parameters and marked increase of central LCs could be demonstrated in SLE patients. SLE alters not only the LC density but also the morphology, modifies corneal homeostasis, and might contribute to the development of dry eye.

  3. Effects of hydroxypyridine derivatives mexidol and emoxypin on the reparative processes in rabbit eye on the models of corneal epithelial defect and conjunctival ischemia.

    Science.gov (United States)

    Chesnokova, N B; Beznos, O V; Pavlenko, T A; Zabozlaev, A A; Pavlova, M V

    2015-01-01

    Deepithelialization of the cornea (diameter 7 mm) was performed in rabbits and the rate of defect epithelialization was evaluated. Conjunctival ischemia was modeled by application of graduated alkaline burn. Antioxidant activity and content of nitrates and nitrites was measured in the tear fluid before and after burn by chemiluminescence and Griess methods, respectively. Emoxypin and mexidol promoted healing of corneal epithelial defect at the stage of epitheliocyte migration to the defect area and at the stage of their proliferation, respectively. After treatment with both agents, the area of conjunctival ischemia decreased more rapidly, but the efficiency of mexidol was higher. Antioxidant activity and content of products of NO metabolism in tear fluid decreased after burn. Mexidol, but not emoxypin, increased these parameters. Thus, mexidol and emoxypin have different effects on corneal epithelialization and conjunctival ischemia and effects of mexidol are more pronounced.

  4. Derivation of corneal endothelial cell-like cells from rat neural crest cells in vitro.

    Directory of Open Access Journals (Sweden)

    Chengqun Ju

    Full Text Available The aim of this study was to investigate the feasibility of inducing rat neural crest cells (NCC to differentiate to functional corneal endothelial cell (CEC-like cells in vitro. Rat NCC were induced with adult CEC-derived conditioned medium. Immunofluorescence, flow cytometry and real time RT-PCR assay were used to detect expression of the corneal endothelium differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2. CFDA SE-labeled CEC-like cells were transplanted to the corneal endothelium of a rat corneal endothelium deficiency model, and an eye-down position was maintained for 24 hours to allow cell attachment. The animals were observed for as long as 2 months after surgery and underwent clinical and histological examination. Spindle-like NCC turned to polygonal CEC-like after induction and expressed N-cadherin, FoxC1, Pitx2, zonula occludens-1 and sodium-potassium pump Na(+/K(+ ATPase. The corneas of the experimental group were much clearer than those of the control group and the mean corneal thickness in the experimental group was significantly less than in the control group7, 14, 21 and 28 days after surgery. Confocal microscopy through focusing and histological analysis confirmed that green fluorescence-positive CEC-like cells formed a monolayer covering the Descemet's membrane in the experimental group. In conclusion, CEC-like cells derived from NCCs displayed characters of native CEC, and the induction protocol provides guidance for future human CEC induction from NCC.

  5. [Protection of corneal endothelium from apoptosis by gene and cell therapy].

    Science.gov (United States)

    Fuchsluger, T A

    2016-06-01

    Protection of corneal endothelium from apoptosis using gene and cell therapy is in a translational phase. This approach offers advantages for eye banking and after transplantation. Safe vehicles for gene or cell therapeutic transduction of corneal endothelium with nucleic acids are available. This strategy will be further developed in consultation with the Paul Ehrlich Institute and European regulatory authorities.

  6. Corneal endothelial cell changes associated with cataract surgery in patients with type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Hugod, Mikkel; Storr-Paulsen, Allan; Norregaard, Jens Christian;

    2011-01-01

    To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation.......To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation....

  7. Effect of the combination of basic fibroblast growth factor and cysteine on corneal epithelial healing after photorefractive keratectomy in patients affected by myopia

    Science.gov (United States)

    Meduri, Alessandro; Scorolli, Lucia; Scalinci, Sergio Zaccaria; Grenga, Pier Luigi; Lupo, Stefano; Rechichi, Miguel; Meduri, Enrico

    2014-01-01

    Background: This study sought to evaluate the effect of basic fibroblast growth factor eye drops and cysteine oral supplements on corneal healing in patients treated with photorefractive keratectomy (PRK). Materials and Methods: One hundred and twenty patients treated bilaterally with PRK for myopia were enrolled at one of two eye centers (Clinica Santa Lucia, Bologna, Italy and Department of Ophthalmology, University of Magna Graecia, Catanzaro, Italy) and were treated at the former center. Sixty patients included in the study group (Group 1) were treated postoperatively with topical basic fibroblast growth factor plus oral L-cysteine supplements, whereas 60 subjects included in the control group (Group 2) received basic fibroblast growth factor eye drops. We recorded the rate of corneal re-epithelialization and patients were followed-up every 30 days for 6 months. Statistical analyses were performed on the collected data. Results: The eyes in Group 1 demonstrated complete re-epithelialization at Day 5, whereas the eyes in Group 2 achieved this status on Day 6. No side-effects were reported. Conclusions: Patients treated with basic fibroblast growth factor eye drops and L-cysteine oral supplements benefit from more rapid corneal re-epithelialization. In human eyes, this combination treatment appeared to be safe and effective in accelerating corneal surfacing after surgery. Financial Disclosure: No author has any financial or proprietary interest in any material or method used in this study. Trial Registration: Current Controlled Trials ISRCTN73824458. PMID:24145571

  8. Effect of the combination of basic fibroblast growth factor and cysteine on corneal epithelial healing after photorefractive keratectomy in patients affected by myopia

    Directory of Open Access Journals (Sweden)

    Alessandro Meduri

    2014-01-01

    Full Text Available Background: This study sought to evaluate the effect of basic fibroblast growth factor eye drops and cysteine oral supplements on corneal healing in patients treated with photorefractive keratectomy (PRK. Materials and Methods: One hundred and twenty patients treated bilaterally with PRK for myopia were enrolled at one of two eye centers (Clinica Santa Lucia, Bologna, Italy and Department of Ophthalmology, University of Magna Graecia, Catanzaro, Italy and were treated at the former center. Sixty patients included in the study group (Group 1 were treated postoperatively with topical basic fibroblast growth factor plus oral L-cysteine supplements, whereas 60 subjects included in the control group (Group 2 received basic fibroblast growth factor eye drops. We recorded the rate of corneal re-epithelialization and patients were followed-up every 30 days for 6 months. Statistical analyses were performed on the collected data. Results: The eyes in Group 1 demonstrated complete re-epithelialization at Day 5, whereas the eyes in Group 2 achieved this status on Day 6. No side-effects were reported. Conclusions : Patients treated with basic fibroblast growth factor eye drops and L-cysteine oral supplements benefit from more rapid corneal re-epithelialization. In human eyes, this combination treatment appeared to be safe and effective in accelerating corneal surfacing after surgery. Financial Disclosure: No author has any financial or proprietary interest in any material or method used in this study. Trial Registration: Current Controlled Trials ISRCTN73824458.

  9. Polarized sorting and trafficking in epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Xinwang Cao; Michal A Surma; Kai Simons

    2012-01-01

    The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain,which are separated by tight junctions.The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules.This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers.Here we review the recent advances in the field of polarized sorting in epithelial cells.We especially highlight the role of lipid rafts in apical sorting.

  10. Morphological changes in corneal endothelial cells after penetrating keratoplasty.

    Science.gov (United States)

    Laing, R A; Sandstrom, M; Berrospi, A R; Leibowitz, H M

    1976-09-01

    Fifteen patients who had had a successful penetrating keratoplasty were photographed with the clinical specular microscope and the resulting endothelial photomicrographs were analyzed. The average endothelial cell area was one to six times larger and the average endothelial cell perimeter was one to 2 1/2 times larger than that of a normal cornea of a subject the same age as the donor. In each corneal graft, endothelial cell areas and perimeters clustered tightly around a mean value, although the mean value for different corneas varied significantly. The thickness and transparency of each graft was normal, indicating that within the observed limits the success of the transplantation procedure did not depend on final endothelial cell size or perimeter.

  11. Coronavirus infection of polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Horzinek, M C; Rottier, P J

    1995-01-01

    Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for

  12. Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof

    Science.gov (United States)

    Linardi, Renata L.; Megee, Susan O.; Mainardi, Sarah R.; Senoo, Makoto; Galantino-Homer, Hannah L.

    2015-01-01

    Background The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. Hypothesis/Objectives To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. Methods Indirect immunofluorescence microscopy and immunoblotting were utilized to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14, and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 (pp63, a marker of ESC to transit-amplifying (TA) cell transition) in epithelial tissues of the foot (haired skin, hoof coronet and hoof lamellae) and the eye (limbus and cornea). Results K14 expression was restricted to the basal layer of epidermal lamellae, and to basal and adjacent suprabasal layers of the haired skin, coronet and corneal limbus. Coronary and lamellar epidermis was negative for both K3 and K10, which were expressed in the cornea/limbus epithelium and haired skin epidermis, respectively. Variable expression of p63 with relatively low to high levels of phosphorylation was detected in individual basal and suprabasal cells of all epithelial tissues examined. Conclusions This is the first report of the characterization of tissue-specific keratin marker expression and the localization of putative epithelial progenitor cell populations, including ESCs (high p63 expression with low pp63 levels) and TA cells (high expression of both p63 and pp63), in the horse. These results will aid further investigation of epidermal and corneal epithelial biology and regenerative therapies in horses. PMID:25963063

  13. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level....... It is concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes....

  14. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level....... It is concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes....

  15. Wound healing of intestinal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Masahiro Iizuka; Shiho Konno

    2011-01-01

    The intestinal epithelial cells (IECs) form a selective permeability barrier separating luminal content from underlying tissues. Upon injury, the intestinal epithelium undergoes a wound healing process. Intestinal wound healing is dependent on the balance of three cellular events;restitution, proliferation, and differentiation of epithelial cells adjacent to the wounded area. Previous studies have shown that various regulatory peptides, including growth factors and cytokines, modulate intestinal epithelial wound healing. Recent studies have revealed that novel factors, which include toll-like receptors (TLRs), regulatory peptides, particular dietary factors, and some gastroprotective agents, also modulate intestinal epithelial wound repair. Among these factors, the activation of TLRs by commensal bacteria is suggested to play an essential role in the maintenance of gut homeostasis. Recent studies suggest that mutations and dysregulation of TLRs could be major contributing factors in the predisposition and perpetuation of inflammatory bowel disease. Additionally, studies have shown that specific signaling pathways are involved in IEC wound repair. In this review, we summarize the function of IECs, the process of intestinal epithelial wound healing, and the functions and mechanisms of the various factors that contribute to gut homeostasis and intestinal epithelial wound healing.

  16. Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration.

    Science.gov (United States)

    Coraux, Christelle; Roux, Jacqueline; Jolly, Thomas; Birembaut, Philippe

    2008-08-15

    In healthy subjects, the respiratory epithelium forms a continuous lining to the airways and to the environment, and plays a unique role as a barrier against external deleterious agents to protect the airways from the insults. In respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), chronic bronchitis, or asthma, the airway epithelium is frequently remodeled and injured, leading to the impairment of its defense functions. The rapid restoration of the epithelial barrier is crucial for these patients. The complete regeneration of the airway epithelium is a complex phenomenon, including not only the epithelial wound repair but also the epithelial differentiation to reconstitute a fully well differentiated and functional epithelium. The regeneration implies two partners: the epithelial stem/progenitor cells and factors able to regulate this process. Among these factors, epithelial cells-extracellular matrix (ECM) interactions play a crucial role. The secretion of a provisional ECM, the cell-ECM relationships through epithelial receptors, and the remodeling of the ECM by proteases (mainly matrix metalloproteinases) contribute not only to airway epithelial repair by modulating epithelial cell migration and proliferation, but also to the differentiation of repairing cells leading to the complete restoration of the wounded epithelium. A better characterization of resident stem cells and of effectors of the regeneration process is an essential prerequisite to propose new regenerative therapeutics to patients suffering from infectious/inflammatory respiratory diseases.

  17. Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

    Institute of Scientific and Technical Information of China (English)

    Shu-Long; Wang; Ge; Zhao; Wei; Zhu; Xiao-Meng; Dong; Ting; Liu; Yuan-Yuan; Li; Wen-Gang; Song; Yi-Qiang; Wang

    2015-01-01

    AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

  18. Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

    Directory of Open Access Journals (Sweden)

    Shu-Long Wang

    2015-01-01

    Full Text Available AIM: To investigate into the potential involvement of pyrin containing 3 gene (NLRP3, a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses. METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1 (HSV-1. Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40 (SV40-immortalized human corneal epithelial cell line were also examined. Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β. RESULTS: The NLRP3 activation induced by HSV-1 infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore, in the SV40-immortalized human corneal epithelial cells, NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium (known as an inhibitor of NLRP3 activation effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot. CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

  19. Use of Corneal Confocal Microscopy to Detect Corneal Nerve Loss and Increased Dendritic Cells in Patients With Multiple Sclerosis.

    Science.gov (United States)

    Bitirgen, Gulfidan; Akpinar, Zehra; Malik, Rayaz A; Ozkagnici, Ahmet

    2017-07-01

    Multiple sclerosis (MS) is characterized by demyelination, axonal degeneration, and inflammation. Corneal confocal microscopy has been used to identify axonal degeneration in several peripheral neuropathies. To assess corneal subbasal nerve plexus morphologic features, corneal dendritic cell (DC) density, and peripapillary retinal nerve fiber layer (RNFL) thickness in patients with MS. This single-center, cross-sectional comparative study was conducted at a tertiary referral university hospital between May 27, 2016, and January 30, 2017. Fifty-seven consecutive patients with relapsing-remitting MS and 30 healthy, age-matched control participants were enrolled in the study. Corneal subbasal nerve plexus measures and DC density were quantified in images acquired with the laser scanning in vivo corneal confocal microscope, and peripapillary RNFL thickness was measured with spectral-domain optical coherence tomography. Corneal nerve fiber density, nerve branch density, nerve fiber length, DC density, peripapillary RNFL thickness, and association with the severity of neurologic disability as assessed by the Kurtzke Expanded Disability Status Scale (score range, 0-10; higher scores indicate greater disability) and Multiple Sclerosis Severity Score (score range, 0.01-9.99; higher scores indicate greater severity). Of the 57 participants with MS, 42 (74%) were female and the mean (SD) age was 35.4 (8.9) years; of the 30 healthy controls, 19 (63%) were female and the mean (SD) age was 34.8 (10.2) years. Corneal nerve fiber density (mean [SE] difference, -6.78 [2.14] fibers/mm2; 95% CI, -11.04 to -2.52; P = .002), nerve branch density (mean [SE] difference, -17.94 [5.45] branches/mm2; 95% CI, -28.77 to -7.10; P = .001), nerve fiber length (mean [SE] difference, -3.03 [0.89] mm/mm2; 95% CI, -4.81 to -1.25; P = .001), and the mean peripapillary RNFL thickness (mean [SE] difference, -17.06 [3.14] μm; 95% CI, -23.29 to -10.82; P < .001) were reduced in patients with MS compared

  20. Platelet response to corneal abrasion is necessary for acute inflammation and efficient re-epithelialization

    Science.gov (United States)

    Purpose: Adhesion molecules play a critical role in leukocyte emigration to wound sites, but differences are evident in different vascular beds. This study investigates the contributions of P-selectin to neutrophil emigration into the cornea following central epithelial abrasion. Methods: Re-epithel...

  1. Corneal Fibroblasts as Sentinel Cells and Local Immune Modulators in Infectious Keratitis

    Directory of Open Access Journals (Sweden)

    Ken Fukuda

    2017-08-01

    Full Text Available The cornea serves as a barrier to protect the eye against external insults including microbial pathogens and antigens. Bacterial infection of the cornea often results in corneal melting and scarring that can lead to severe visual impairment. Not only live bacteria but also their components such as lipopolysaccharide (LPS of Gram-negative bacteria contribute to the development of inflammation and subsequent corneal damage in infectious keratitis. We describe the important role played by corneal stromal fibroblasts (activated keratocytes as sentinel cells, immune modulators, and effector cells in infectious keratitis. Corneal fibroblasts sense bacterial infection through Toll-like receptor (TLR–mediated detection of a complex of LPS with soluble cluster of differentiation 14 (CD14 and LPS binding protein present in tear fluid. The cells then initiate innate immune responses including the expression of chemokines and adhesion molecules that promote the recruitment of inflammatory cells necessary for elimination of the infecting bacteria. Infiltrated neutrophils are activated by corneal stromal collagen and release mediators that stimulate the production of pro–matrix metalloproteinases by corneal fibroblasts. Elastase produced by Pseudomonas aeruginosa (P. aeruginosa activates these released metalloproteinases, resulting in the degradation of stromal collagen. The modulation of corneal fibroblast activation and of the interaction of these cells with inflammatory cells and bacteria is thus important to minimize corneal scarring during treatment of infectious keratitis. Pharmacological agents that are able to restrain such activities of corneal fibroblasts without allowing bacterial growth represent a potential novel treatment option for prevention of excessive scarring and tissue destruction in the cornea.

  2. Epithelial cell-cell junctions and plasma membrane domains

    NARCIS (Netherlands)

    Giepmans, Ben N. G.; van Ijzendoorn, Sven C. D.

    Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally,

  3. Epithelial cell-cell junctions and plasma membrane domains

    NARCIS (Netherlands)

    Giepmans, Ben N. G.; van Ijzendoorn, Sven C. D.

    2009-01-01

    Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally, comp

  4. Epithelial Cell Apoptosis and Lung Remodeling

    Institute of Scientific and Technical Information of China (English)

    Kazuyoshi Kuwano

    2007-01-01

    Lung epithelium is the primary site of lung damage in various lung diseases. Epithelial cell apoptosis has been considered to be initial event in various lung diseases. Apoptosis signaling is classically composed of two principle pathways. One is a direct pathway from death receptor ligation to caspase cascade activation and cell death. The other pathway triggered by stresses such as drugs, radiation, infectious agents and reactive oxygen species is mediated by mitochondria. Endoplasmic reticulum has also been shown to be the organelle to mediate apoptosis.Epithelial cell death is followed by remodeling processes, which consist of epithelial and fibroblast activation,cytokine production, activation of coagulation pathway, neoangiogenesis, re-epithelialization and fibrosis.Epithelial and mesenchymal interaction plays important roles in these processes. Further understanding of apoptosis signaling and its regulation by novel strategies may lead to effective treatments against various lung diseases. We review the recent advances in the understanding of apoptosis signaling and discuss the involvement of apoptosis in lung remodeling.

  5. Mesenchymal stem cells: Potential role in corneal wound repair and transplantation

    Institute of Scientific and Technical Information of China (English)

    Fei; Li; Shao-Zhen; Zhao

    2014-01-01

    Corneal diseases are a major cause of blindness in the world. Although great progress has been achieved in the treatment of corneal diseases, wound healing after severe corneal damage and immunosuppressive therapy after corneal transplantation remain prob-lematic. Mesenchymal stem cells(MSCs) derived from bone marrow or other adult tissues can differentiate into various types of mesenchymal lineages, such as osteocytes, adipocytes, and chondrocytes, both in vivo and in vitro. These cells can further differentiate into specific cell types under specific conditions. MSCs migrate to injury sites and promote wound healing by secreting anti-inflammatory and growth factors. In ad-dition, MSCs interact with innate and acquired immune cells and modulate the immune response through their powerful paracrine function. Over the last decade, MSCs have drawn considerable attention because of their beneficial properties and promising therapeutic prospective. Furthermore, MSCs have been applied to various studies related to wound healing, autoim-mune diseases, and organ transplantation. This review discusses the potential functions of MSCs in protecting corneal tissue and their possible mechanisms in corneal wound healing and corneal transplantation.

  6. Preservation of Ocular Epithelial Limbal Stem Cells: The New Frontier in Regenerative Medicine.

    Science.gov (United States)

    Lužnik, Zala; Bertolin, Marina; Breda, Claudia; Ferrari, Barbara; Barbaro, Vanessa; Schollmayer, Petra; Ferrari, Stefano

    2016-01-01

    Significant advances have been made in the field of ocular regenerative medicine. Promising stem cell-based therapeutic strategies have been translated into the clinical practice over the last few decades. These new stem cell-based therapies offer the possibility of permanently restoring corneal epithelium in patients with severe disabling and blinding ocular surface disease. The European Union has already classified stem cell-based therapies as "medicinal products". Therefore, manipulation is strictly regulated according to the defined conditions of good manufacturing practice, with the production of stem cell therapeutics at only accredited production sites authorized by the national regulatory agencies. In this regard, as first medical products are licensed for commercial use in Europe enabling a more widespread access to a stem cell-based therapy, the need for safe, validated and reproducible techniques for ex vivo cultured tissue preservation and distribution are coming to the forefront of research. However, these provide various new challenges for biobanking industry such as the retention of viability, good functionality of stem cells and sterility issues. This chapter provides an overview of the current advances in the field of corneal/limbal epithelial stem cell culture preservation techniques using either hypothermic storage or cryopreservation methods, that were used in different culturing steps (from stem cell isolation to the ex vivo epithelial graft preparation), with the reported impact on the post-thawing product recovery.

  7. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  8. Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)

    Science.gov (United States)

    Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko

    2012-01-01

    Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD. PMID:23818740

  9. Limbal melanocytes support limbal epithelial stem cells in 2D and 3D microenvironments.

    Science.gov (United States)

    Dziasko, Marc A; Tuft, Stephen J; Daniels, Julie T

    2015-09-01

    Human limbal epithelial stem cells (LESCs) are essential for the maintenance of the corneal epithelium of the ocular surface. LESCs are located within limbal crypts between the palisades of Vogt in the limbus; the interface between the peripheral cornea and conjunctiva. The limbal crypts have been proposed as a LESC niche owing to their support of epithelial cells, which can form holoclone colonies in vitro. Closely associated with the limbal crypts is a concentrated population of melanocytes. The anatomical location and close proximity to putative LESC suggests that melanocytes might play a role in maintenance of these stem cells in the niche. The aim of this study was to assess the ability of human limbal melanocytes (hLM) to support the expansion of human limbal epithelial cells (LECs) in vitro as an indicator of functional cell-cell interaction. After observing that hLM co-localize with clusters of compact epithelial cells in the native limbal crypts, hLM were isolated from crypt-rich cadaveric limbal biopsies and used as feeders for the culture of LECs. Interestingly, LECs grown on mitotically active hLM were able to generate large epithelial colonies that contained small and compact cells with morphological stem cell characteristics. Immunocytochemistry revealed that LECs expanded on hLM were positive for the expression of the putative stem cell markers CK15, Bmi-1 and p63α and negative for the marker of terminal cell differentiation CK3. LECs and hLM were finally co-cultured on RAFT (real architecture for 3D tissue) collagen tissue equivalents. In 3D co-cultures, hLM promoted multi-layering of the epithelial sheet in which basal cells were maintained in an undifferentiated state. Taken together, these observations suggest melanocytes could play an important role in the maintenance of LESCs in the native human limbal stem cell niche.

  10. Application value of corneal limbus stem cells transplantation to treat pterygium

    Directory of Open Access Journals (Sweden)

    Miao-Ying Zhang

    2017-02-01

    Full Text Available AIM: To analyze the application value of corneal limbus stem cells transplantation in the treatment of pterygium. METHODS: Totally 300 patients(318 eyeswith pterygium were divided into 2 groups according to treatment methods. Patients of the control group(70 patients with 77 eyeswere given simple surgical resection, while patients of the observation group(230 patients with 241 eyeswere given corneal limbus stem cells transplantation after surgical resection. The postoperative recurrent rate, corneal epithelium incision healing time, corneal epithelium healing degree, pain score and tear film break-up time were observed. RESULTS: The corneal epithelium incision healing time of the observation group and control group were respectively 5.2±1.9d and 6.4±1.7d, and the difference had statistical significance(PPP>0.05. At 2wk after treatment, the corneal epithelium healing degree of the observation group was better, and the pain score of the observation group was lower than those of the control group, the differences between groups had statistical significance(PP>0.05. At 1 and 2wk after treatment, the tear film break-up time of the observation group was longer than that of the control group(PCONCLUSION: Corneal limbus stem cell transplantation can prevent postoperative recurrence of pterygium effectively, and its postoperative tissue repair effect is superior to simple surgery.

  11. Effect of Mitomycin-C augmented trabeculectomy on corneal endothelial cells

    Directory of Open Access Journals (Sweden)

    Reza Zarei

    2015-01-01

    Conclusion: MMC application in trabeculectomy seems to cause a small but significant corneal endothelial loss. Most of the damage occurs intraoperatively, or in the early postoperative period, however progressive endothelial cell loss is not a major concern.

  12. Tissue engineering of corneal stromal layer with dermal fibroblasts: phenotypic and functional switch of differentiated cells in cornea.

    Science.gov (United States)

    Zhang, Yan Qing; Zhang, Wen Jie; Liu, Wei; Hu, Xiao Jie; Zhou, Guang Dong; Cui, Lei; Cao, Yilin

    2008-02-01

    Previously, we successfully engineered a corneal stromal layer using corneal stromal cells. However, the limited source and proliferation potential of corneal stromal cells has driven us to search for alternative cell sources for corneal stroma engineering. Based on the idea that the tissue-specific environment may alter cell fate, we proposed that dermal fibroblasts could switch their phenotype to that of corneal stromal cells in the corneal environment. Thus, dermal fibroblasts were harvested from newborn rabbits, seeded on biodegradable polyglycolic acid (PGA) scaffolds, cultured in vitro for 1 week, and then implanted into adult rabbit corneas. After 8 weeks of implantation, nearly transparent corneal stroma was formed, with a histological structure similar to that of its native counterpart. The existence of cells that had been retrovirally labeled with green fluorescence protein (GFP) demonstrated the survival of implanted cells. In addition, all GFP-positive cells that survived expressed keratocan, a specific marker for corneal stromal cells, and formed fine collagen fibrils with a highly organized pattern similar to that of native stroma. However, neither dermal fibroblast-PGA construct pre-incubated in vitro for 3 weeks nor chondrocyte-PGA construct could form transparent stroma. The results demonstrated that neonatal dermal fibroblasts could switch their phenotype in the new tissue environment under restricted conditions. The functional restoration of corneal transparency using dermal fibroblasts suggests that they could be an alternative cell source for corneal stroma engineering.

  13. Lipid polarity and sorting in epithelial cells

    NARCIS (Netherlands)

    van Meer, G.; Simons, K.

    1988-01-01

    Apical and basolateral membrane domains of epithelial cell plasma membranes possess unique lipid compositions. The tight junction, the structure separating the two domains, forms a diffusion barrier for membrane components and thereby prevents intermixing of the two sets of lipids. The barrier appar

  14. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    Science.gov (United States)

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M.; Pluth, Janice M.; Cucinotta, Francis A.

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

  15. Protons sensitize epithelial cells to mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    Minli Wang

    Full Text Available Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1-mediated epithelial-mesenchymal transition (EMT, a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu and hTERT- immortalized human esophageal epithelial cells (EPC were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1 kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1.

  16. Pirfenidone nanoparticles improve corneal wound healing and prevent scarring following alkali burn.

    Directory of Open Access Journals (Sweden)

    Sushovan Chowdhury

    Full Text Available PURPOSE: To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn. METHODS: Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA synthesis by TGFβ induced primary corneal fibroblast cells was evaluated by immunoblotting and immunocytochemistry. Pirfenidone loaded poly (lactide-co-glycolide (PLGA nanoparticles were prepared, characterized and their cellular entry was examined in primary corneal fibroblast cells by fluorescence microscopy. Alkali burn was induced in one eye of Sprague Dawley rats followed by daily topical treatment with free pirfenidone, pirfenidone nanoparticles or vehicle. Corneal re-epithelialization was assessed daily by flourescein dye test; absence of stained area indicated complete re-epithelialization and the time for complete re-epithelialization was determined. Corneal haze was assessed daily for 7 days under slit lamp microscope and graded using a standard method. After 7 days, collagen I deposition in the superficial layer of cornea was examined by immunohistochemistry. RESULTS: Pirfenidone prevented (P<0.05 increase in TGF β induced collagen I and α-SMA synthesis by corneal fibroblasts in a dose dependent manner. Pirfenidone could be loaded successfully within PLGA nanoparticles, which entered the corneal fibroblasts within 5 minutes. Pirfenidone nanoparticles but not free pirfenidone significantly (P<0.05 reduced collagen I level, corneal haze and the time for corneal re-epithelialization following alkali burn. CONCLUSION: Pirfenidone decreases collagen synthesis and prevents myofibroblast formation. Pirfenidone nanoparticles improve corneal wound healing and prevent fibrosis. Pirfenidone nanoparticles are of potential value in treating corneal chemical burns and other corneal fibrotic diseases.

  17. Adipose-derived mesenchymal stem cell administration does not improve corneal graft survival outcome.

    Directory of Open Access Journals (Sweden)

    Sherezade Fuentes-Julián

    Full Text Available The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical

  18. IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Roni M. Shtein

    2012-01-01

    Full Text Available Purpose. To determine time course of effect of lipopolysaccharide (LPS on production of interleukin-8 (IL-8 and monocyte chemotactic protein (MCP by cultured human corneal stromal cells. Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student's t-test. Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05 after corneal stromal cell stimulation with LPS. Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

  19. Investigation the Porous Collagen-Chitosan /Glycosaminoglycans for Corneal Cell Culture as Tissue Engineering Scaffold

    Institute of Scientific and Technical Information of China (English)

    LI Qin-Hua; CHEN Jian-Su

    2005-01-01

    The objective of this study was to produce the porous collagen-chitosan/Glycosanminglycans (GAG) for corneal ceil-seed implant as a three-dimensional tissue engineering scaffold to improve the regeneration corneas. The effect of various content of glycerol as form porous agent to collagen-chitosan/GAG preserved a porous dimensional structure was investigated. The heat-drying was used to prepare porous collagen-chitosan /GAG scaffold. The pore morphology of collagenchitosan/GAG was controlled by changing the concentration of glycerol solution and drying methods. The porous structure morphology was observed by SEM. The diameter of the pores form 10 to 50 μm. The highly porous scaffold had interconnecting pores. The corneal cell morphology was observed under the light microscope. These results suggest that collagen-chitosan/GAG showed that corneal cell have formed confluent layers and resemble the surface of normal corneal cell surface.

  20. [Neurotrophic keratopathy--studies on substance P and the clinical significance of corneal sensation].

    Science.gov (United States)

    Nishida, T; Nakamura, M; Konma, T; Ofuji, K; Nagano, K; Tanaka, T; Enoki, M; Reid, T W; Brown, S M; Murphy, C J; Mannis, M J

    1997-12-01

    Neurotrophic keratopathy, which often follows damage to the trigeminal nerve, is clinically characterized by various types of epithelial disorders and melting of corneal stroma. To understand both the pathology of neurotrophic keratopathy and the physiological significance of corneal sensation, we investigated both the cellular and molecular functions of a sensory neurotransmitter, substance P, in corneal epithelial cells. Our findings prompted us to try a new mode of treatment for neurotrophic keratopathy. Substance P, a member of the tachykinin family, is an 11-amino-acid peptide. In an organ culture system using rabbit corneas, substance P alone had no effect on corneal epithelial migration. In the presence of insulin-like growth factor-1 (IGF-1), however, substance P synergistically facilitated corneal epithelial migration in proportion to the concentration of substance P or of IGF-1. Other neurotransmitters (acetylcholine, norepinephrine, serotonin etc.) or tachykinins (neurokinin A, eledoisin etc.) did not show this synergistic effect with IGF-1. Among receptors for the tachykinin family (NK-1, NK-2, or NK-3) only the NK-1 receptor system was involved in the synergistic effect of substance P and IGF-1 on corneal epithelial migration. IGF-1 affected neither the binding constant nor the number of sites of substance P receptors in corneal epithelial cells, suggesting that the synergistic effect was not regulated at the receptor level. Various extracellular signals activate the intracellular signal transduction system, thus amplifying specific biological functions. We found that the addition of inhibitors of protein kinase C or tyrosine kinase clearly inhibited the synergistic effect of substance P and IGF-1 on corneal epithelial migration, demonstrating that protein kinase C and tyrosine kinase are involved in the synergistic effect. During corneal epithelial wound healing, epithelial cells must attach to a provisional, extracellular fibronectin matrix. We

  1. Airway epithelial cells: current concepts and challenges.

    Science.gov (United States)

    Crystal, Ronald G; Randell, Scott H; Engelhardt, John F; Voynow, Judith; Sunday, Mary E

    2008-09-15

    The adult human bronchial tree is covered with a continuous layer of epithelial cells that play a critical role in maintaining the conduit for air, and which are central to the defenses of the lung against inhaled environmental concomitants. The epithelial sheet functions as an interdependent unit with the other lung components. Importantly, the structure and/or function of airway epithelium is deranged in major lung disorders, including chronic obstructive pulmonary disease, asthma, and bronchogenic carcinoma. Investigations regarding the airway epithelium have led to many advances over the past few decades, but new developments in genetics and stem cell/progenitor cell biology have opened the door to understanding how the airway epithelium is developed and maintained, and how it responds to environmental stress. This article provides an overview of the current state of knowledge regarding airway epithelial stem/progenitor cells, gene expression, cell-cell interactions, and less frequent cell types, and discusses the challenges for future areas of investigation regarding the airway epithelium in health and disease.

  2. The graft of autologous adipose-derived stem cells in the corneal stromal after mechanic damage.

    Directory of Open Access Journals (Sweden)

    Xiao-Yun Ma

    Full Text Available This study was designed to explore the feasibility of using autologous rabbit adipose derived stem cells (rASCs as seed cells and polylactic-co-glycolic acid (PLGA as a scaffold for repairing corneal stromal defects. rASCs isolated from rabbit nape adipose tissue were expanded and seeded on a PLGA scaffold to fabricate cell-scaffold constructs. After 1 week of cultivation in vitro, the cell-scaffold complexes were transplanted into corneal stromal defects in rabbits. In vivo, the autologous rASCs-PLGA constructed corneal stroma gradually became transparent without corneal neovascularization after 12 weeks. Hematoxylin and eosin staining and transmission electron microscopy examination revealed that their histological structure and collagen fibril distribution at 24 weeks after implantation were similar to native counterparts. As to the defect treated with PLGA alone, the stromal defects remained. And scar tissue was observed in the untreated-group. The implanted autologous ASCs survived up to 24 weeks post-transplantation and differentiated into functional keratocytes, as assessed by the expression of aldehyde-3-dehydrogenase1A1 (ALDH1A1 and cornea-specific proteoglycan keratocan. Our results revealed that autologous rASCs could be one of the cell sources for corneal stromal restoration in diseased corneas or for tissue engineering of a corneal equivalent.

  3. Isolation of epithelial cells with hepatobiliary phenotype.

    Science.gov (United States)

    Castorina, Sergio; Luca, Tonia; Torrisi, Antonella; Privitera, Giovanna; Panebianco, Mariangela

    2008-01-01

    The regenerative capacity of the liver after partial hepatectomy or chemical injury is well known. In human liver, the resident progenitor cells are called "hepatic progenitor cells" (HPCs) while the term "oval cells" should be discouraged in order to indicate the stem cell compartment. The aim of our study was first to analyse the cellular aspects of liver regeneration through differentiation in cholangiocytes and hepatocytes, and then to characterise resident progenitor cells, using "primary cultured hepatocytes" derived from healthy adult human livers. Human hepatocytes were isolated from fresh surgical specimens of patients who underwent hepatic resections in our Clinical Centre surgery operating room. Hepatic differentiation and function were analysed by immunocytochemistry techniques and the presence of liver epithelial cell populations within normal adult human liver, was demonstrated by immunohistochemistry analysis. These cells expanded in vitro and showed the capacity for self-renewal and multipotent differentiation. Human liver stem cells expressed several mesenchymal markers, such as CD44, but not haematopoietic stem cell markers. In addition, these cells expressed alpha-fetoprotein, albumin, CK7 and CK19, indicating a partial commitment to hepatic and biliary cells. Interestingly the expression of both hepatocytes and biliary markers in HPCs reflects the bipotential nature of the hepatic stem cells toward both the hepatic and biliary lineage. According to their immature and bipotential phenotype, hepatic epithelial cells might represent a pool of precursors in the healthy human adult liver.

  4. The human airway epithelial basal cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Neil R Hackett

    Full Text Available BACKGROUND: The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. METHODOLOGY/PRINCIPAL FINDINGS: Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. CONCLUSION/SIGNIFICANCE: The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of

  5. Reversible transdifferentiation of alveolar epithelial cells.

    Science.gov (United States)

    Danto, S I; Shannon, J M; Borok, Z; Zabski, S M; Crandall, E D

    1995-05-01

    Alveolar epithelial type II (AT2) cells have been thought to be the progenitors of terminally differentiated type I (AT1) cells in the adult animal in vivo. In this study, we used an AT1 cell-specific monoclonal antibody (mAb VIII B2) to investigate expression of the AT1 cell phenotype accompanying reversible changes in expression of the AT2 cell phenotype. AT2 cells were isolated and cultured either on attached collagen gels or on gels detached 1 or 4 days after plating and maintained thereafter as floating gels. Monolayers on both attached and floating gels were harvested on days 4 and 8 and analyzed by electron microscopy for changes in morphology and binding of mAb VIII B2. Results indicate that: (1) alveolar epithelial cells (AEC) on attached gels develop characteristics of the AT1 cell phenotype, (2) AEC on gels detached on day 1 maintain features of the AT2 cell phenotype (and do not react with mAb VIII B2), and (3) the expression of AT1 cell phenotypic traits seen by day 4 on attached gels is reversed after detachment. We conclude that commitment to the AT1 and AT2 cell lineages requires continuous regulatory input to maintain the differentiated states, and that transdifferentiation between AT2 and AT1 cells may be reversible.

  6. Corneal wound healing is compromised by immunoproteasome deficiency.

    Directory of Open Access Journals (Sweden)

    Deborah A Ferrington

    Full Text Available Recent studies have revealed roles for immunoproteasome in regulating cell processes essential for maintaining homeostasis and in responding to stress and injury. The current study investigates how the absence of immunoproteasome affects the corneal epithelium under normal and stressed conditions by comparing corneas from wildtype (WT mice and those deficient in two immunoproteasome catalytic subunits (lmp7(-/-/mecl-1(-/-, L7M1. Immunoproteasome expression was confirmed in WT epithelial cells and in cells of the immune system that were present in the cornea. More apoptotic cells were found in both corneal explant cultures and uninjured corneas of L7M1 compared to WT mice. Following mechanical debridement, L7M1 corneas displayed delayed wound healing, including delayed re-epithelialization and re-establishment of the epithelial barrier, as well as altered inflammatory cytokine production compared to WT mice. These results suggest that immunoproteasome plays an important role in corneal homeostasis and wound healing.

  7. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  8. Envisaging an allogenic Corneal endothelial precursor/Stem Cell Bank (CESBANK

    Directory of Open Access Journals (Sweden)

    Parikumar P

    2008-01-01

    Full Text Available Bullous Keratopathy (BK affects thousands of people in India every year. Though in early stages it is manageable medically, advanced disease warrants either total corneal transplantation or partial thickness transplantation for which a donor-cadaver cornea is necessary. Amano et al have reported the successful treatment of BK in animal models using in-vitro expanded human corneal endothelial precursors; though the rabbits had to be kept facing eye down to allow gravity assisted settling of the cells to the summit of the cornea where the damage had been created. For successful treatment using the above method, a human being has to lie prone with eyes immobilized for 24-36 Hrs. This is extremely discomforting and hence not practical. Corneal endothelium removed from the button and transported at varying temperature conditions for 48Hrs was successfully cultured in NCRM and this was reported earlier. We are working on a suitable scaffold to retain the cells in situ until their attachment to the damaged portion of the corneal endothelium enabling it to heal without the patient having to lie prone. With such capability, we envisage to make a corneal endothelial precursor/stem cell (CES bank named as CESBANK to make in-vitro expanded CES available for patients with corneal diseases, most commonly Bullous Keratopathy (BK.

  9. N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering

    Directory of Open Access Journals (Sweden)

    Bernadette K. Madathil

    2014-01-01

    Full Text Available Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.

  10. Progenitors for the Corneal Endothelium and Trabecular Meshwork: A Potential Source for Personalized Stem Cell Therapy in Corneal Endothelial Diseases and Glaucoma

    Directory of Open Access Journals (Sweden)

    Wing Yan Yu

    2011-01-01

    Full Text Available Several adult stem cell types have been found in different parts of the eye, including the corneal epithelium, conjunctiva, and retina. In addition to these, there have been accumulating evidence that some stem-like cells reside in the transition area between the peripheral corneal endothelium (CE and the anterior nonfiltering portion of the trabecular meshwork (TM, which is known as the Schwalbe's Ring region. These stem/progenitor cells may supply new cells for the CE and TM. In fact, the CE and TM share certain similarities in terms of their embryonic origin and proliferative capacity in vivo. In this paper, we discuss the putative stem cell source which has the potential for replacement of lost and nonfunctional cells in CE diseases and glaucoma. The future development of personalized stem cell therapies for the CE and TM may reduce the requirement of corneal grafts and surgical treatments in glaucoma.

  11. Cultivation of corneal endothelial cells on a pericellular matrix prepared from human decidua-derived mesenchymal cells.

    Directory of Open Access Journals (Sweden)

    Ryohei Numata

    Full Text Available The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na⁺/K⁺-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

  12. Cultivation of corneal endothelial cells on a pericellular matrix prepared from human decidua-derived mesenchymal cells.

    Science.gov (United States)

    Numata, Ryohei; Okumura, Naoki; Nakahara, Makiko; Ueno, Morio; Kinoshita, Shigeru; Kanematsu, Daisuke; Kanemura, Yonehiro; Sasai, Yoshiki; Koizumi, Noriko

    2014-01-01

    The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs) is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM) as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs) via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na⁺/K⁺-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

  13. Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells.

    Science.gov (United States)

    Kramerov, Andrei A; Saghizadeh, Mehrnoosh; Ljubimov, Alexander V

    2016-04-07

    The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

  14. Primary corneal papilloma and squamous cell carcinoma associated with pigmentary keratitis in four dogs.

    Science.gov (United States)

    Bernays, M E; Flemming, D; Peiffer, R L

    1999-01-15

    Squamous cell carcinoma (SCC) and squamous papilloma are rarely reported as primary lesions of the cornea in dogs. One case of corneal papilloma and 3 cases of SCC, each arising as a primary central corneal neoplasm rather than spreading from adjacent limbal conjunctiva, were reviewed. The most common cause of SCC in animals is chronic exposure of lightly pigmented epithelium to UV light; however, all dogs in this study had a history of chronic pigmentary keratitis. Three of the 4 dogs were of brachycephalic breeds with naturally proptotic eyes and oversized palpebral fissures that may have exposed the cornea to greater excessive solar radiation. Alternatively, mechanical factors that caused chronic changes in the cornea may have been causative factors for induction of primary dysplastic or neoplastic changes. Primary corneal neoplasia should be considered in the differential diagnosis of corneal masses.

  15. IκB kinase β regulates epithelium migration during corneal wound healing.

    Directory of Open Access Journals (Sweden)

    Liang Chen

    Full Text Available The IKKβ is known to regulate transcription factor NF-κB activation leading to inflammatory responses. Recent gene knockout studies have shown that IKKβ can orchestrate local inflammatory responses and regulate homeostasis of epithelial tissues. To investigate whether IKKβ has an intrinsic role in epithelial cells, we established an in vivo system in the immune privileged corneal epithelium. We generated triple transgenic Krt12(rtTA/rtTAt/tet-O-Cre/Ikkβ(F/F (Ikkβ(ΔCE/ΔCE mice by crossing the Krt12-rtTA knock-in mice, which express the reverse tetracycline transcription activator in corneal epithelial cells, with the tet-O-Cre and Ikkβ(F/F mice. Doxycycline-induced IKKβ ablation occurred in corneal epithelial cells of triple transgenic Ikkβ(ΔCE/ΔCE mice, but loss of IKKβ did not cause ocular abnormalities in fetal development and postnatal maintenance. Instead, loss of IKKβ significantly delayed healing of corneal epithelial debridement without affecting cell proliferation, apoptosis or macrophage infiltration. In vitro studies with human corneal epithelial cells (HCEpi also showed that IKKβ was required for cytokine-induced cell migration and wound closure but was dispensable for cell proliferation. In both in vivo and in vitro settings, IKKβ was required for optimal activation of NF-κB and p38 signaling in corneal epithelial cells, and p38 activation is likely mediated through formation of an IKKβ-p38 protein complex. Thus, our studies in corneal epithelium reveal a previously un-recognized role for IKKβ in the control of epithelial cell motility and wound healing.

  16. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells.

    Science.gov (United States)

    Simon, R H; DeHart, P D; Todd, R F

    1986-11-01

    The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing.

  17. Central corneal abscess.

    Science.gov (United States)

    van Bijsterveld, O P

    1976-05-01

    Central corneal abscess developed in the experimental animal after inoculation of biologically active staphylococcal strains in a paracentral epithelial lesion of the cornea. These abscesses did not ulcerate, developed only with high inocula, occurred more frequently in immunized rabbits. A serpiginous type of ulceration did not develop at the site of the initial epithelial lesion nor at any other place in the cornea. Histologically, the lesions consisted of densely packed polymorphonuclear leukocytes between the corneal lamellae.

  18. Intestinal epithelial cells in inflammatory bowel diseases

    Institute of Scientific and Technical Information of China (English)

    Giulia; Roda; Alessandro; Sartini; Elisabetta; Zambon; Andrea; Calafiore; Margherita; Marocchi; Alessandra; Caponi; Andrea; Belluzzi; Enrico; Roda

    2010-01-01

    The pathogenesis of inflammatory bowel diseases (IBDs) seems to involve a primary defect in one or more of the elements responsible for the maintenance of intestinal homeostasis and oral tolerance. The most important element is represented by the intestinal barrier, a complex system formed mostly by intestinal epithelial cells (IECs). IECs have an active role in producing mucus and regulating its composition; they provide a physical barrier capable of controlling antigen traff ic through the intestinal muco...

  19. Bevacizumab application delays epithelial healing in rabbit cornea.

    Science.gov (United States)

    Kim, Tae-im; Chung, Jae Lim; Hong, Jin Pyo; Min, Kyung; Seo, Kyoung Yul; Kim, Eung Kweon

    2009-10-01

    Vascular endothelial growth factor (VEGF) is essential for neovascularization, but the use of anti-VEGF therapies to inhibit neovascularization may influence epithelial wound healing. Here, the effects of bevacizumab on corneal epithelial wound healing time in rabbit models, cell proliferation, and expression of integrins in human corneal epithelial and fibroblast cells were evaluated. To compare epithelial wound healing times, epithelial defect sizes were measured after application of bevacizumab topical eye drops at 0, 0.5, 1.0, 1.5, 2.5, or 5 mg/mL, twice daily, to mechanically debrided epithelia of rabbit corneas. The cellular covering of wounded areas and expression of Ki67 were assessed after scrape injuries in cultures of human corneal epithelial and fibroblast cells. Expression of cell surface integrins and collagens was measured using plates coated with mouse monoclonal antibodies against human adhesion molecules, and relevant mRNA levels were assessed by reverse-transcription-polymerase chain reaction (RT-PCR). The application of bevacizumab topical eye drops at 1.0, 1.5, 2.5, or 5 mg/mL delayed rabbit corneal epithelial healing. Cell cultures growing under high concentrations of bevacizumab showed delay in the proliferation of corneal epithelial and fibroblast cells. Surface expression of mRNA encoding integrins and collagens were decreased by 1.5 mg/mL of bevacizumab. Bevacizumab delayed corneal epithelial wound healing and inhibited integrin expression. When bevacizumab is used to reduce the development of new corneal vessels, slight delays in epithelial wound healing are possible and cellular proliferation is to be expected.

  20. Long-term corneal endothelial cell changes in pediatric intraocular lens reposition and exchange cases.

    Science.gov (United States)

    Wang, Yan; Wu, Mingxing; Zhu, Liyuan; Liu, Yizhi

    2012-04-01

    To evaluate long-term corneal endothelial cell changes of intraocular lens (IOL) reposition and exchange in children. State key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China In this retrospective study, all IOL reposition and exchange procedures performed in patients under 14 years old between January 1999 and April 2009 were included. Follow-up outcomes included corneal endothelial cell density, hexagonality, coefficient of variance, average cell size. IOL reposition procedures in 12 eyes (12 cases) (reposition group, RPG), and IOL exchanges in eight eyes (eight cases) (exchange group, EXG) were performed because of IOL pupillary capture or IOL dislocation. Median of follow-up was 44.5 months in RPG and 66.2 months in EXG. The density of corneal endothelial cells in RPG (2,053 ± 493/mm(2)) and EXG (2,100 ± 758/mm(2)) was significantly decreased in comparison to the control eyes (3,116 ± 335/mm(2)). Hexagonality of corneal endothelial cells and coefficient of variance showed no difference among the control group, RPG and EXG (P > 0.05). The density of corneal endothelial cells was conspicuously decreased after IOL reposition or exchange procedures in childhood cases. Longer follow-up must be conducted in these cases.

  1. Multiple endpoint analysis of BAC-preserved and unpreserved antiallergic eye drops on a 3D-reconstituted corneal epithelial model

    OpenAIRE

    Pauly, A.; Brasnu, E.; Riancho, L.; Brignole-Baudouin, F.; Baudouin, C

    2011-01-01

    Purpose To compare the effects of benzalkonium chloride (BAC)-preserved and unpreserved antiallergic eye drops on the human 3D-reconstituted corneal epithelial model (3D-HCE). Methods 3D-HCE were treated for 24 h followed or not by a 24 h post-incubation recovery period (24 h+24 h) with phosphate-buffered saline (PBS), 0.01% BAC, unpreserved formulations of ketotifen, N Acetyl-Aspartyl Glutamic Acid (NAAGA), cromoglycate, or BAC-preserved commercial formulations of ketotifen, olopatadine, epi...

  2. Survival and integration of tissue-engineered corneal stroma in a model of corneal ulcer.

    Science.gov (United States)

    Zhang, Chao; Nie, Xin; Hu, Dan; Liu, Yuan; Deng, Zhihong; Dong, Rui; Zhang, Yongjie; Jin, Yan

    2007-08-01

    Tissue-engineered replacement of diseased or damaged tissue has become a reality for some types of tissue, such as skin and cartilage. Tissue-engineered corneal stroma represents a promising concept to overcome the limitations of cornea replacement with allograft. In this study, porcine cornea was decellularized by a series of extraction methods, and the in vivo biocompatibility of the scaffold was measured subcutaneously in rabbits (n = 8). These were not acutely rejected and no abscesses were observed by hematoxylin and eosin staining at the 8th week, indicating that the scaffolds had good biocompatibility. To investigate the potential value of clinical applications, rabbit stromal keratocytes were implanted onto decellularized scaffolds to fabricate tissue-engineered corneal stroma. Allograft, tissue-engineered corneal stroma, or scaffolds were implanted into a model of corneal ulcer. The survival and reconstruction of corneal transplantation were morphologically evaluated by light and electron microscopy until the 32nd week after implantation. Experiments involving transplantation indicated that the epithelial and stromal defect healed quickly, with improvement in corneal clarity. The integration of the graft was accompanied by neurite ingrowth from the host tissue. By 16 weeks after transplantation, the cornea had gradually regained an intact state similar to that of normal cornea. Our results demonstrate that the tissue-engineered corneal stroma with allogenetic cells is a promising therapeutic method for corneal injury.

  3. Hypercalcemia Leads to Delayed Corneal Wound Healing in Ovariectomized Rats.

    Science.gov (United States)

    Nagai, Noriaki; Ogata, Fumihiko; Kawasaki, Naohito; Ito, Yoshimasa; Funakami, Yoshinori; Okamoto, Norio; Shimomura, Yoshikazu

    2015-01-01

    Hypercalcemia is often observed in postmenopausal women as well as in patients with primary hyperparathyroidism or malignant tumors. In this study, we investigated the relationship between calcium ion (Ca(2+)) levels in lacrimal fluid and the rate of corneal wound healing in hypercalcemia using ovariectomized (OVX) rat debrided corneal epithelium. We also determined the effects of Ca(2+) levels on cell adhesion, proliferation and viability in a human cornea epithelial cell line (HCE-T). The calcium content in bones of OVX rats decreased after ovariectomy. Moreover, the Ca(2+) content in the blood of OVX rats was increased 1 month after ovariectomy, and decreased. The Ca(2+) content in the lacrimal fluid of OVX rats was also increased after ovariectomy, and then decreased similarly as in blood. Corneal wound healing in OVX rats was delayed in comparison with Sham rats (control rats), and a close relationship was observed between the Ca(2+) levels in lacrimal fluid and the rate of corneal wound healing in Sham and OVX rats (y=-0.7863x+8.785, R=0.78, n=25). In addition, an enhancement in Ca(2+) levels caused a decrease in the viability in HCE-T cells. It is possible that enhanced Ca(2+) levels in lacrimal fluid may cause a decrease in the viability of corneal epithelial cells, resulting in a delay in corneal wound healing. These findings provide significant information that can be used to design further studies aimed at reducing corneal damage of patients with hypercalcemia.

  4. Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus.

    Science.gov (United States)

    Fan, TingJun; Zhao, Jun; Fu, YongFeng; Cong, RiShan; Guo, RuiChao; Liu, WanShun; Han, BaoQin; Yu, QiuTao; Wang, Jing

    2007-04-01

    To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium containing chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sulfate at 37 degrees C, 5% CO(2). The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to confluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical researches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.

  5. Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium con- taining chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sul- fate at 37℃, 5% CO2. The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to con- fluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical re- searches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.

  6. Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

    Institute of Scientific and Technical Information of China (English)

    FAN TingJun; ZHAO Jun; FU YongFeng; CONG RiShan; GUO RuiChao; LIU WanShun; HAN BaoQin; YU QiuTao; WANG Jing

    2007-01-01

    To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium containing chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride,culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sulfate at 37℃, 5% CO2. The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to confluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical researches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.

  7. Treatment of corneal squamous cell carcinoma using topical 1% 5-fluorouracil as monotherapy.

    Science.gov (United States)

    Dorbandt, Daniel M; Driskell, Elizabeth A; Hamor, Ralph E

    2016-05-01

    The purpose of this report is to discuss the use of topical 1% 5-fluorouracil as a sole therapy for canine corneal squamous cell carcinoma (SCC). A 12-year-old castrated male pug was evaluated for a well-demarcated, central, 3 mm in diameter, pale pink, raised, right corneal mass. An incisional biopsy was obtained using a #64 beaver blade after topical anesthesia and without sedation. A definitive diagnosis of corneal SCC was obtained after histopathologic evaluation of the biopsy. Topical 1% 5-fluorouracil ointment was applied to the right eye four times daily for 2 weeks followed by no treatment for 2 weeks, then treatment again twice daily for 2 weeks. The cornea remained free of recurrence 10 months after cessation of treatment. In dogs affected with corneal SCC, topical 1% 5-fluorouracil monotherapy may be a viable and cost-effective treatment option with minimal side effects. This chemotherapy agent may also have an effect on corneal pigmentation. Chronic cyclosporine therapy did not contribute to the pathogenesis of corneal SCC in the case described.

  8. Characterization of Human Mammary Epithelial Stem Cells

    Science.gov (United States)

    2010-10-01

    breast is highly expressed by luminal epithelial cells and is less expressed by basal cells19,20. In contrast, CD49f (a6 integrin) has an inverse pattern...mouse stretched on its back. The hose and nose cone from the anesthetic vaporizer are securely attached to one side of the plate, and a heated pad is...the mouse by a nose cone. Check that the mouse has reached surgical anesthesia by loss of pedal withdrawal reflex . ! cautIon Institutional review

  9. [Morphometric changes of corneal endothelial cells in pseudoexfoliation syndrome and pseudoexfoliation glaucoma].

    Science.gov (United States)

    de Juan-Marcos, L; Cabrillo-Estévez, L; Escudero-Domínguez, F A; Sánchez-Jara, A; Hernández-Galilea, E

    2013-11-01

    To evaluate the corneal endothelial morphometry and central corneal thickness (CCT) in pseudoexfoliative (PEX) eyes with and without glaucoma and to compare with normal eyes and eyes with primary open-angle glaucoma (POAG). A total of 166 patients were included in this study: 36 eyes with pseudoexfoliation syndrome (PXS), 30 eyes with pseudoexfoliation glaucoma (PXG), 40 eyes with POAG, and 60 normal eyes. Corneal endothelial cell density (ECD), coefficient of variation (CV) in cell size, and percentage of hexagonal cells, were measured using a non-contact specular microscope, whereas CCT was measured with an ultrasonic pachymeter. ECD and percentage of hexagonal cells were lower in PEX groups and in the POAG group compared with normal eyes, while the CV in cell size was greater. There was a tendency for greater cell loss and morphological abnormalities of the corneal endothelial cells in PXG eyes compared to PXS eyes, when all pseudoexfoliative eyes were analyzed together. Changes in endothelial cells increased with age. There were no significant differences in mean CCT between the four groups. Endothelial cell density is significantly decreased, and pleomorphism and polymegathism of cells are increased in PEX eyes, particularly when intraocular pressure is high. Copyright © 2012 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  10. Lactobacillus decelerates cervical epithelial cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Katarina Vielfort

    Full Text Available We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.

  11. Confocal microscopy and electrophysiological study of single patient corneal endothelium cell cultures

    Science.gov (United States)

    Tatini, Francesca; Rossi, Francesca; Coppi, Elisabetta; Magni, Giada; Fusco, Irene; Menabuoni, Luca; Pedata, Felicita; Pugliese, Anna Maria; Pini, Roberto

    2016-04-01

    The characterization of the ion channels in corneal endothelial cells and the elucidation of their involvement in corneal pathologies would lead to the identification of new molecular target for pharmacological treatments and to the clarification of corneal physiology. The corneal endothelium is an amitotic cell monolayer with a major role in preserving corneal transparency and in regulating the water and solute flux across the posterior surface of the cornea. Although endothelial cells are non-excitable, they express a range of ion channels, such as voltage-dependent Na+ channels and K+ channels, L-type Ca2 channels and many others. Interestingly, purinergic receptors have been linked to a variety of conditions within the eye but their presence in the endothelium and their role in its pathophysiology is still uncertain. In this study, we were able to extract endothelial cells from single human corneas, thus obtaining primary cultures that represent the peculiarity of each donor. Corneas were from tissues not suitable for transplant in patients. We characterized the endothelial cells by confocal microscopy, both within the intact cornea and in the primary endothelial cells cultures. We also studied the functional role of the purinergic system (adenosine, ATP and their receptors) by means of electrophysiological recordings. The experiments were performed by patch clamp recordings and confocal time-lapse microscopy and our results indicate that the application of purinergic compounds modulates the amplitude of outward currents in the isolated endothelial cells. These findings may lead to the proposal of new therapies for endothelium-related corneal diseases.

  12. Aligned Fibrous Scaffold Induced Aligned Growth of Corneal Stroma Cells in vitro Culture

    Institute of Scientific and Technical Information of China (English)

    GAO Yan; YAN Jing; CUI Xue-jun; WANG Hong-yan; WANG Qing

    2012-01-01

    To investigate the contribution of fibre arrangement to guiding the aligned growth of corneal stroma cells,aligned and randomly oriented fibrous scaffolds of gelatin and poly-L-lactic acid(PLLA) were fabricated by electrospinning.A comparative study of two different systems with corneal stroma cells on randomly organized and aligned fibres were conducted.The efficiency of the scaffolds for inducing the aligned growth of cells was assessed by morphological observation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT) assay.Results show that the cells cultured on both randomly oriented and aligned scaffolds maintained normal morphology and well spreading as well as long term proliferation.Importantly,corneal stroma cells grew high orderly on the aligned scaffold,while the cells grew disordered on the randomly oriented scaffold.Moreover,the cells exhibited higher viability in aligned scaffold than that in randomly oriented scaffold.These results indcate that electrospinng to prepare aligned fibrous scaffolds has provided an effective approach to the aligned growth of corneal stroma cells in vitro.Our findings that fiber arrangement plays a crucial role in guiding the aligned growth of cells may be helpful to the development of better biomaterials for tissue engineered cornea.

  13. Nanoscale topography-induced modulation of fundamental cell behaviors of rabbit corneal keratocytes, fibroblasts, and myofibroblasts.

    Science.gov (United States)

    Pot, Simon A; Liliensiek, Sara J; Myrna, Kathern E; Bentley, Ellison; Jester, James V; Nealey, Paul F; Murphy, Christopher J

    2010-03-01

    Keratocyte-to-myofibroblast differentiation is a key factor in corneal wound healing. The purpose of this study was to determine the influence of environmental nanoscale topography on keratocyte, fibroblast, and myofibroblast cell behavior. Primary rabbit corneal keratocytes, fibroblasts, and myofibroblasts were seeded onto planar polyurethane surfaces with six patterned areas, composed of anisotropically ordered grooves and ridges with a 400-, 800-, 1200-, 1600-, 2000-, and 4000-nm pitch (pitch = groove + ridge width). After 24 hours cells were fixed, stained, imaged, and analyzed for cell shape and orientation. For migration studies, cells on each patterned surface were imaged every 10 minutes for 12 hours, and individual cell trajectories and migration rates were calculated. Keratocytes, fibroblasts, and myofibroblasts aligned and elongated to pitch sizes larger than 1000 nm. A lower limit to the topographic feature sizes that the cells responded to was identified for all three phenotypes, with a transition zone around the 800- to 1200-nm pitch size. Fibroblasts and myofibroblasts migrated parallel to surface ridges larger than 1000 nm but lacked directional guidance on submicron and nanoscale topographic features and on planar surfaces. Keratocytes remained essentially immobile. Corneal stromal cells elongated, aligned, and migrated, differentially guided by substratum topographic features. All cell types failed to respond to topographic features approximating the dimensions of individual stromal fibers. These findings contribute to our understanding of corneal stromal cell biology in health and disease and their interaction with biomaterials and their native extracellular matrix.

  14. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair;

    2003-01-01

    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

  15. A comparison of three methods for trephining donor corneal buttons: endothelial cell loss and microscopic ultrastructural evaluation.

    Science.gov (United States)

    Moshirfar, Majid; Meyer, Jay J; Kang, Paul C

    2009-11-01

    To evaluate the ultrastructure of the cut edge and associated endothelial cell loss following donor cornea trephination with a standard punch, vacuum punch, and vacuum trephine and artificial anterior chamber system. This laboratory investigation compared trephinations (8.0 mm) performed on human corneas using either a standard posterior punch (n = 12), vacuum posterior punch (n = 12), or vacuum trephine and artificial anterior chamber system (n = 12). Specular microscopy was performed before and after trephination to determine central endothelial cell density. Light and scanning electron microscopy were performed to evaluate the structure of the trephined edge. Endothelial cell-free distances from the trephinated edges were measured on light microscopy sections. Central endothelial cell loss (cells/mm(2)) after trephination was -14.0 +/- 49.9 (SD) for the standard posterior punch, -85.6 +/- 87.0 for the vacuum posterior punch, -116.0 +/- 223.1 for the vacuum trephine and artificial anterior chamber system. Endothelial cell-free distances from the trephined margin were 63 +/- 22 microm, 85 +/- 13 microm, and 123 +/- 48 microm for the three respective methods. The edges of grafts cut with anterior trephination were inward sloping from the epithelial to endothelial surfaces, while both posterior punches created outward sloping edges. Increased fibrillar disruption at edges was seen following anterior trephination. Different trephination methods produce distinct cut morphologies with the anterior trephination approach, resulting in more irregular margins. The anterior approach was associated with increased variability and greater endothelial cell loss than the studied posterior approaches. The use of corneal scissors may contribute to the morphologic features of the corneal button seen following anterior trephination.

  16. Discovery of molecular markers to discriminate corneal endothelial cells in the human body.

    Directory of Open Access Journals (Sweden)

    Masahito Yoshihara

    Full Text Available The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

  17. Discovery of molecular markers to discriminate corneal endothelial cells in the human body.

    Science.gov (United States)

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji

    2015-01-01

    The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

  18. Effects of Phthalates on the Human Corneal Endothelial Cell Line B4G12

    DEFF Research Database (Denmark)

    Krüger, Tanja; Cao, Yi; Kjærgaard, Søren K.;

    2012-01-01

    Phthalates are industrial chemicals used in many cosmetics. We evaluated an in vitro model for eye irritancy testing using the human corneal endothelial cell line B4G12. Cell proliferation and toxicity were assessed after exposing to di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2...

  19. Effects of phacoemulsification on the intraocular pressure and corneal endothelial cells of the patients with glaucoma

    Institute of Scientific and Technical Information of China (English)

    Ye Zhao; Zhi-Feng Liu

    2016-01-01

    Objective:To explore the intraocular pressure and corneal endothelial cells integrity changes in cataract phacoemulsification after anti-glaucoma surgery.Methods:Phacoemulsification was performed in 102 patients (118 eyes) with cataract after anti-glaucoma surgery and the intraocular pressure and corneal endothelial cell integrity changes of patients were observed at Day 1 and 3, first week and first month before and after surgery, including central corneal endothelial cell density, average cell area (AVE), cell area of coefficient of variation (CV) and central corneal thickness (CCT).Results:The intraocular pressure was elevated, the central endothelial cell density was reduced, the AVE, the CV and CTT thicken were increased at Day 1 and 3, first week and first month after surgery. The difference compared with preoperative was statistically significant. The intraocular pressure and CTT almost recovered to preoperative levels in 1 month after cataract phacoemulsification and the difference was not statistically significant; while the central endothelial cell density was still decreased and AVE and CV were still increased and the difference of these indexes and the coefficient of the patients was statistically significant compared with before surgery.Conclusions:For the patients with anti-glaucoma after cataract phacoemulsification, intraocular pressure and endothelial cell integrity change was initially observed at Day 1 after surgery, whereas they can almost return to the preoperative level in a month after surgery.

  20. Cell reintegration: Stray epithelial cells make their way home.

    Science.gov (United States)

    Wilson, Tyler J; Bergstralh, Dan T

    2017-06-01

    Ongoing work shows that misplaced epithelial cells have the capacity to reintegrate back into tissue layers. This movement appears to underlie tissue stability and may also control aspects of tissue structure. A recent study reveals that cell reintegration in at least one tissue, the Drosophila follicular epithelium, is based on adhesion molecules that line lateral cell surfaces. In this article we will review these observations, discuss their implications for epithelial tissue development and maintenance, and identify future directions for study. © 2017 WILEY Periodicals, Inc.

  1. Value of human amniotic epithelial cells in tissue engineering for cornea.

    Science.gov (United States)

    Fatimah, Simat Siti; Ng, Sook Luan; Chua, Kien Hui; Hayati, Abdul Rahman; Tan, Ay Eeng; Tan, Geok Chin

    2010-11-01

    Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.

  2. Confocal microscopy of epithelial and langerhans cells of the cornea in patients using travoprost drops containing two different preservatives.

    Science.gov (United States)

    Marsovszky, László; Resch, Miklós D; Visontai, Zsuzsanna; Németh, János

    2014-07-01

    The recently developed confocal cornea microscopy offers the opportunity to examine pathologies of the cornea and to gain insight into the activity of innate immunity. We aimed to investigate the corneal epithelial and Langerhans cell (LC) densities along with dry eye parameters in primary open-angle glaucoma (POAG) subjects, treated with either of two commercially available travoprost 0.004 % topical medications containing different preservatives. (1: benzalkonium chloride 0.015 % (TravBAK) and 2: polyquaternium-1 (PQ) 0.001 % (TravPQ). Consecutive case series of nineteen POAG patients on TravBAK (mean age: 64.8 ± 13.6 years), nineteen POAG patients on TravPQ (mean age: 66.8 ± 11.3 years) and nineteen age-matched healthy control subjects (63.8 ± 8.2 years). Ocular surface disease index (OSDI), lid parallel conjunctival folds (LIPCOF), Schirmer test (ST) and tear break up time (TBUT) were assessed, and then corneal epithelial and LC densities were investigated with confocal microscopy. Tear production was significantly reduced in both glaucoma patient groups compared to healthy individuals (p < 0.05). TBUT was significantly reduced and epithelial cell densities were significantly greater in patients treated with TravBAK compared to healthy individuals (p < 0.05 for all). LC densities were greater in both glaucoma groups compared to control subjects (p < 0.05 for all). Travoprost therapy may compromise ocular surface. The limited alertness of the corneal immune system found in patients with TravPQ can be considered as indicators of a less disturbed ocular surface and better controlled corneal homeostasis.

  3. RECURRENT CORNEAL EROSION SYNDROME (a review

    Directory of Open Access Journals (Sweden)

    S. V. Trufanov

    2015-01-01

    Full Text Available Recurrent corneal erosion (RCE syndrome is characterized by episodes of recurrent spontaneous epithelial defects. Main clinical symptoms (pain, redness, photophobia, lacrimation occurred at night. Corneal lesions revealed by slit lamp exam vary depending on the presence of corneal epithelium raise, epithelial microcysts or epithelial erosions, stromal infiltrates and opacities. Microtraumas, anterior corneal dystrophies, and herpesvirus give rise to RCE. Other causes or factors which increase the risk of RCE syndrome include meibomian gland dysfunction, keratoconjunctivitis sicca, diabetes, and post-LASIK conditions. Basal membrane abnormalities and instability of epithelial adhesion to stroma play a key role in RCE pathogenesis. Ultrastructural changes in RCE include abnormalities of basal epithelial cells and epithelial basal membrane, absence or deficiency of semi-desmosomes, loss of anchor fibrils. Increase in matrix metalloproteinases and collagenases which contribute to basal membrane destruction results in recurrent erosions and further development of abnormal basal membrane. The goals of RCE therapy are to reduce pain (in acute stage, to stimulate re-epithelization, and to restore «adhesion complex» of basal membrane. In most cases, RCE responds to simple conservative treatment that includes lubricants, healing agents, and eye patches. RCEs that are resistant to simple treatment, require complex approach. Non-invasive methods include long-term contact lens use, instillations of autologous serum (eye drops, injections of botulinum toxin (induces ptosis, antiviral agent use or oral intake of metalloproteinase inhibitors. Cell membrane stabilizers, i.e., antioxidants, should be included into treatment approaches as well. Antioxidant effect of Emoxipine promotes tissue reparation due to the prevention of cell membrane lipid peroxidation as well as due to its anti-hypoxic, angioprotective, and antiplatelet effects. If conservative therapy

  4. Cells of Origin of Epithelial Ovarian Cancers

    Science.gov (United States)

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0280 TITLE: Cells of Origin of Epithelial Ovarian Cancers PRINCIPAL INVESTIGATOR: Zhe Li, PhD CONTRACTING...Xie, Zhe Li 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail: zli4@rics.bwh.harvard.edu 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND...Lined Inclusion Cysts or Teratomas. PLoS ONE 8, e65067. Sherman-Baust, C.A., Kuhn, E., Valle, B.L., Shih Ie, M., Kurman, R.J., Wang , T.L., Amano, T

  5. p53 protein expression in corneal squamous cell carcinomas of dogs

    Directory of Open Access Journals (Sweden)

    Lucas Bahdour Cossi

    2015-06-01

    Full Text Available Ocular tumors play an increasing concern in veterinary ophthalmology. Corneal squamous cell carcinoma is unfrequent in dogs, and by this way it has little studies. Studies that investigated the carcinogenesis mechanisms wich could help to the development of ocular squamous cell carcinoma (SCC in dog are rare. The aim of this work was to identify by immunohistochemical techniques, the p53 protein expression in the spontaneous dog corneal SCC. For this work, were used five cases of corneal SCC and one case of actinic keratitis. The sections were obtained from paraffin-wax blocks and submitted to histopathological and immunohistochemical analysis. All the six samples showed immunolabeling to cytokeratin and p53 protein. These results support the conclusions that the immunoreactivity of p53 protein by immunohistochemistry is present in canine corneal SCC suppporting its role in carcinogenesis of this tumor, but not provides prognostic indicators in cases of SCC corneal in dog; and can be a association of exposure to solar radiation with the possible mutation of the TP53 gene.

  6. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells.

    OpenAIRE

    Simon, R H; DeHart, P D; Todd, R F

    1986-01-01

    The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neut...

  7. Cultivation of Human Microvascular Endothelial Cells on Topographical Substrates to Mimic the Human Corneal Endothelium

    Directory of Open Access Journals (Sweden)

    Jie Shi Chua

    2013-03-01

    Full Text Available Human corneal endothelial cells have a limited ability to replicate in vivo and in vitro. Allograft transplantation becomes necessary when an accident or trauma results in excessive cell loss. The reconstruction of the cornea endothelium using autologous cell sources is a promising alternative option for therapeutic or in vitro drug testing applications. The native corneal endothelium rests on the Descemet’s membrane, which has nanotopographies of fibers and pores. The use of synthetic topographies mimics the native environment, and it is hypothesized that this can direct the behavior and growth of human microvascular endothelial cells (HMVECs to resemble the corneal endothelium. In this study, HMVECs are cultivated on substrates with micron and nano-scaled pillar and well topographies. Closely packed HMVEC monolayers with polygonal cells and well-developed tight junctions were formed on the topographical substrates. Sodium/potassium (Na+/K+ adenine triphosphatase (ATPase expression was enhanced on the microwells substrate, which also promotes microvilli formation, while more hexagonal-like cells are found on the micropillars samples. The data obtained suggests that the use of optimized surface patterning, in particular, the microtopographies, can induce HMVECs to adopt a more corneal endothelium-like morphology with similar barrier and pump functions. The mechanism involved in cell contact guidance by the specific topographical features will be of interest for future studies.

  8. Characterization of protocadherin-1 expression in primary bronchial epithelial cells : association with epithelial cell differentiation

    NARCIS (Netherlands)

    Koning, Henk; Sayers, Ian; Stewart, Ceri E.; de Jong, Debora; ten Hacken, Nick H. T.; Postma, Dirkje S.; van Oosterhout, Antoon J. M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2012-01-01

    Protocadherin-1 (PCDH1) is a novel susceptibility gene for asthma that is expressed in airway epithelium. We aimed to characterize PCDH1 mRNA transcripts and protein expression in primary bronchial epithelial cells and to determine regulation of PCDH1 during mucociliary differentiation. Total RNA an

  9. Transplantation of reconstructed corneal layer composed of corneal epithelium and fibroblasts on a lyophilized amniotic membrane to severely alkali-burned cornea.

    Science.gov (United States)

    Jang, In-Keun; Ahn, Jae-Il; Shin, Jun-Seop; Kwon, Young-Sam; Ryu, Yang-Hwan; Lee, Jeong-Kyu; Park, Jung-Keug; Song, Kye-Yong; Yang, Eun-Kyung; Kim, Jae-Chan

    2006-06-01

    The purpose of this article was to evaluate the graft efficacy of reconstructed corneal layer, composed of autologous corneal epithelium and fibroblasts on a lyophilized amniotic membrane (LAM), in a severely alkali-burned corneal model. After biopsy specimens were obtained from the left eyes of 24 rabbits, the corneal epithelial cells and fibroblasts were expanded in vitro and the corneal layer was reconstructed on LAM. Thirty-six eyes of rabbits underwent alkali burn (1 N NaOH, 30 s) to create a limbal deficiency and a deeply damaged corneal stroma. Four weeks later, group 1 underwent a graft of the reconstructed corneal layer composed of autologous corneal epithelium and fibroblasts on LAM. Group 2 was transplanted with a graft of the reconstructed autologous corneal epithelium, and group 3 served as a control without surgery. Wound healing and stabilization of the ocular surfaces occurred much faster in group 1 than in groups 2 and 3. The eyes in group 3 revealed typical limbal deficiencies with conjuctivalization and persistent corneal epithelial defects. However, the corneas in group 1 developed only mild peripheral neovascularization. Immunohistochemical staining in group 1 demonstrated that p63, cytokeratin 3, E-cadherin, transforming growth factor (TGF)-beta1, and collagen IV were expressed strongly in the corneal epithelium and basement membrane. On the basis of these results, transplantation of the reconstructed corneal layer, composed of autologous corneal epithelium and fibroblasts on LAM, partially accelerated the recovery of the alkali-injured rabbit ocular surface, and might be useful therapeutically for the treatment of patients with severely damaged cornea.

  10. Primary Adult Human Retinal Pigment Epithelial Cell Cultures on Human Amniotic Membranes

    Directory of Open Access Journals (Sweden)

    Singhal Shweta

    2005-01-01

    Full Text Available Purpose: Retinal pigment epithelial (RPE cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM as substrate. Materials and Methods: Human cadaver eyeballs (16 were obtained from the eye bank after corneal trephination. RPE cells were harvested by a mechanical dissection of the inner choroid surface (10, group 1 or by b enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2. The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students′ ′t′ test. Results: Eleven samples (68.75% showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days in the mechanical group, significantly earlier ( P Conclusions: Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes . Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion

  11. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  12. Nuclear microscopy of rat colon epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, M., E-mail: phyrenmq@nus.edu.sg [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Rajendran, Reshmi [Lab of Molecular Imaging, Singapore Bioimaging Consotium, 11 Biopolis Way, 02-02 Helios, Singapore 138667 (Singapore); Ng, Mary [Department of Pharmacology, National University of Singapore (Singapore); Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Jenner, Andrew Michael [Illawara Health and Medical Research Institute (IHMRI), University of Wollongong, NSW 2522 (Australia)

    2011-10-15

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  13. Nuclear microscopy of rat colon epithelial cells

    Science.gov (United States)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  14. Pterygium is related to a decrease in corneal endothelial cell density.

    Science.gov (United States)

    Hsu, Min-Yen; Lee, Hsin-Nung; Liang, Chiao-Ying; Wei, Li-Chen; Wang, Chun-Yuan; Lin, Keng-Hung; Shen, Ying-Cheng

    2014-07-01

    The aim of this study was to investigate the relationship between pterygium and a decrease in the corneal endothelial cell density (ECD) in patients with unilateral primary pterygium. In this retrospective cross-sectional study, 90 consecutive patients with unilateral primary pterygium were enrolled from January 2010 to June 2012. Corneal ECD was measured in both eyes, and the fellow eyes were considered as controls. The relationship between the percentage of pterygium to cornea and a decrease in the ECD was analyzed. An increase in astigmatism in eyes with pterygium was evaluated for association with decreased ECD using the Pearson correlation test. The percentage of pterygium to cornea ranged from 3.5% to 65.2%, with a median of 12.35%. The difference in the corneal ECD between eyes with pterygium and control eyes ranged from +9.6% to -37.7%, with a median of -9.75%. The results of the Pearson correlation statistical test showed a strong logarithmic correlation between a decrease in the corneal ECD and the percentage of pterygium to cornea (R = 0.688, P decrease in the ECD in eyes with pterygium. Pterygium is related to a decrease in corneal ECD. Surgical intervention should be considered in patients with extensive pterygium involvement in the cornea or a significant increase in astigmatism.

  15. Rethinking immunological privilege: implications for corneal and limbal stem cell transplantation.

    Science.gov (United States)

    Williams, K A; Coster, D J

    1997-11-01

    Immunological privilege operates within the normal eye by multiple passive and active mechanisms, including antigen sequestration, maintenance of an immunosuppressive local environment and induction of apoptotic death in infiltrating cells of the immune system. Ocular privilege might have developed to protect the eye from the collateral damage associated with an inflammatory response to invading pathogens. Nevertheless, corneal grafts do undergo irreversible immunological rejection and, furthermore, corneal graft rejection is very similar at a histological level to the rejection processes that operate in vascularized organ grafts. Ocular privilege is thus relative. The question arises as to how corneal grafts are rejected in the face of so many mechanisms designed to prevent immune responses from operating inside the eye--a question that is still essentially unanswered.

  16. CORNEAL STROMAL THINNING: A RARE CORNEAL COMPLICATION AFTER BARE SCL ERA PTERYG I UM EXCISION TECHNIQUE

    Directory of Open Access Journals (Sweden)

    Sulaiman Abdul

    2015-06-01

    Full Text Available INTRODUCTION : Bare sclera technique without using any anti - mitotic drugs are commonly employed in rural population. Corneal dellen formation and recurrence of pterygium are more common in these cases. But the corneal complication like stromal thinning, necrosis of corne a and sclera are not common. The corneal epithelium is a highly differentiated cell type that is self - renewing. Also corneal epithelium is important for the stromal replacement in the situations like chemical, thermal burns, ocular surgery like pterygium s urgery. Interference with status of stem cell replacement and as a consequent to it, stromal thinning is occurring in the pterygium surgery. P atient 1: A 68 yr s. old male patient underwent pterygium surgery ( B aresclera excision technique. After 30 days he developed corneal thinning with the punched out partial stromal loss without perforation or descmetocele or scleral thinning. Patient 2: A 60yr s. old male patient underwent pterygium surgery 2months back, he developed same type of corneal thinning. Both P atients were treated with tear drops and improved. CONCLUSION : The stromal thinning in these two cases is may be due to chemical factors like collagenase which might have been released from the traumatised conjunctival epithelial cells causing thinning wit hout replacement of stroma by limbal stem cells.

  17. Effect of pirfenidone on the proliferation of rat corneal stromal cells

    Directory of Open Access Journals (Sweden)

    Jun-Jie Chen

    2015-02-01

    Full Text Available AIM: To investigate the effects of pirfenidone(PFDon the proliferation and transfomring growth factor-β1(TGF-β1expression in vitro culture rat corneal stromal cells. METHODS: Corneal stromal cells from 8 to 10wk SD rats were isolated, cultured and treated with different concentrations of PFD 0mg/mL(control group, 0.15mg/mL(experimental group Ⅰ, 0.3mg/mL(experimental group Ⅱ, 1mg/mL(experimental group Ⅲfor 48h. CCK-8 assay was performed to assess cell proliferation, while immunocytochemistry and Western Blot were used to detect the expression of ki-67 and TGF-β1 expression, respectively. RESULTS: Compared with control group, PFD significantly inhibited the proliferation in a dose-dependent manner(all P1 in a dose-dependent manner(PCONCLUSION: Pirfenidone can significantly inhibit the proliferation of rat corneal stromal cell by down regulating TGF-β1 expression, therefore, it has potential prospect in lightening the corneal wound healing reaction.

  18. EFFECT OF HUMAN AMNIOTIC MEMBRANE ON CORNEAL EPITHELIUM AND YAC-1 CELL

    Institute of Scientific and Technical Information of China (English)

    叶纹; 沈玺; 钟一声

    2003-01-01

    Objective To study the effect of the amniotic membrane on enhancing the proliferation of corneal epithelia and YAC 1 cell.MethodsAfter the primary culture of the rabbits corneal epithelia and YAC 1 cells, they were seeded on the upper surface or stromal matrix side of amniotic membrane respectively. The proliferation results were observed by MTT test.ResultsThe amniotic membrane was found significantly enhancing the proliferation of corneal epithelia on the d1,d3,and d5 after culture. The proliferation rate was 28.93%,23.32%,23.41%(P<0.05)respectively, but the d7 proliferation rate was 20.72%(P>0.05).On the d1,d3,d7 after culture,the YAC 1 cells proliferation rate was 34.87%,36.28%,33.86%(P<0.01)respectively.ConclusionOur results demonstrated that the amniotic membrane could enhance the prolifera tion of both corneal epithelia and YAC 1 cells significantly. Although amniotic membrane has been suggested as an ideal material for reconstruction of ocular surface, special attention should be paid during amniotic membrane transplantation for treating ocular surface lesion resulted from epibulbar tumors.

  19. Uranium induces apoptosis in lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Periyakaruppan, Adaikkappan; Sarkar, Shubhashish; Sadanandan, Bindu; Thomas, Renard; Wilson, Bobby L. [Texas Southern University, Environmental Toxicology Program, Department of Chemistry, Houston, TX (United States); Ravichandran, Prabakaran; Sharma, Chidananda S.; Ramesh, Vani; Hall, Joseph C.; Ramesh, Govindarajan T. [Norfolk State University, Molecular Toxicology Laboratory, Department of Biology, Center for Biotechnology and Biomedical Sciences, Norfolk, VA (United States)

    2009-06-15

    Uranium is a naturally occurring radioactive material present everywhere in the environment. It is toxic because of its chemical or radioactive properties. Uranium enters environment mainly from mines and industry and cause threat to human health by accumulating in lungs as a result of inhalation. In our previous study, we have shown the effectiveness of antioxidant system response to the oxidative stress induced by uranyl acetate (UA) in rat lung epithelial (LE) cells. As part of our continuing studies; here, we investigated the mechanism underlying when LE cells are exposed to different concentration of UA. Oxidative stress may lead to apoptotic signaling pathways. LE cells treated with 0.25, 0.5 and 1 mM of UA results in dose and time-dependent increase in activity of both caspases-3 and -8. Increase in the concentration of cytochrome-c oxidase in cytosol was seen in LE cells treated with 1 mM UA as a result of mitochondria membrane permeability. The cytochrome-c leakage may trigger the apoptotic pathway. TUNEL assay performed in LE cells treated with 1 mM of UA showed significant incorporation of dNTPs in the nucleus after 24 h. In the presence of the caspase inhibitors, we observed the significant decrease in the activity of caspases-8 and -3 in 0.5 and 1 mM UA-treated LE cells. (orig.)

  20. Mechanical stretch triggers rapid epithelial cell division through Piezo1.

    Science.gov (United States)

    Gudipaty, S A; Lindblom, J; Loftus, P D; Redd, M J; Edes, K; Davey, C F; Krishnegowda, V; Rosenblatt, J

    2017-03-02

    Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.

  1. Progressive transformation of immortalized esophageal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Zhong-Ying Shen; Li-Yan Xu; Min-Hua Chen; Jian Shen; Wei-Jia Cai; Yi Zeng

    2002-01-01

    .CONCLUSION: In continual cultivation of fetal esophageal epithelial cells with transduction of HPV18E6E7, cells from the 10th to the 85th passage were changed gradually from preimmortal, immortal, precancerous to malignantly transformed stages. All of these changes were in a dynamic progressive process. The establishment of a continuous line of esophageal epithelium may provide a in vitro model of carcinogenesis induced by HPV.

  2. Globoside accelerates the differentiation of dental epithelial cells into ameloblasts

    Institute of Scientific and Technical Information of China (English)

    Takashi Nakamura; Yuta Chiba; Masahiro Naruse; Kan Saito; Hidemitsu Harada; Satoshi Fukumoto

    2016-01-01

    Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside (Gb4), a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins (including neurotrophin-4) and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts.

  3. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    Science.gov (United States)

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

  4. Potential uses of milk epithelial cells: a review

    OpenAIRE

    2002-01-01

    International audience; Secretions collected from the mammary gland of different species contain heterogeneous populations of cells including lymphocytes, neutrophils, macrophages and epithelial cells in different species. Several factors influence the somatic cell count in milk and the distribution of cell types, such as species, infection status, physiological status and management practices. The epithelial cells are shed into milk during the lactation process. Most of them are viable and e...

  5. Desmosome dynamics in migrating epithelial cells requires the actin cytoskeleton

    Science.gov (United States)

    Roberts, Brett J.; Pashaj, Anjeza; Johnson, Keith R.; Wahl, James K.

    2011-01-01

    Re-modeling of epithelial tissues requires that the cells in the tissue rearrange their adhesive contacts in order to allow cells to migrate relative to neighboring cells. Desmosomes are prominent adhesive structures found in a variety of epithelial tissues that are believed to inhibit cell migration and invasion. Mechanisms regulating desmosome assembly and stability in migrating cells are largely unknown. In this study we established a cell culture model to examine the fate of desmosomal components during scratch wound migration. Desmosomes are rapidly assembled between epithelial cells at the lateral edges of migrating cells and structures are transported in a retrograde fashion while the structures become larger and mature. Desmosome assembly and dynamics in this system are dependent on the actin cytoskeleton prior to being associated with the keratin intermediate filament cytoskeleton. These studies extend our understanding of desmosome assembly and provide a system to examine desmosome assembly and dynamics during epithelial cell migration. PMID:21945137

  6. In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins.

    Science.gov (United States)

    Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Khang, Gilson; Soker, Shay

    2013-02-01

    The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation.

  7. Observing planar cell polarity in multiciliated mouse airway epithelial cells.

    Science.gov (United States)

    Vladar, Eszter K; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type.

  8. Thermosensitive chitosan-based hydrogels for sustained release of ferulic acid on corneal wound healing.

    Science.gov (United States)

    Tsai, Ching-Yao; Woung, Lin-Chung; Yen, Jiin-Cherng; Tseng, Po-Chen; Chiou, Shih-Hwa; Sung, Yen-Jen; Liu, Kuan-Ting; Cheng, Yung-Hsin

    2016-01-01

    Oxidative damage to cornea can be induced by alkaline chemical burn which may cause vision loss or blindness. Recent studies showed that exogenous application of natural antioxidants may be a potential treatment for corneal wound healing. However, low ocular bioavailability and short residence time are the limiting factors of topically administered antioxidants. Ferulic acid (FA) is a natural phenolic compound and an excellent antioxidant. The study was aimed to investigate the effects of FA in corneal epithelial cells (CECs) under oxidative stress and evaluate the feasibility of use the thermosensitive chitosan-based hydrogel containing FA for corneal wound healing. The results demonstrated that post-treatment of FA on CECs could decrease the inflammation-level and apoptosis. In the rabbit corneal alkali burn model, post-treatment FA-loaded hydrogel may promote the corneal wound healing. The results of study suggest that FA-loaded hydrogel may have the potential applications in treating corneal alkali burn.

  9. Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells.

    Science.gov (United States)

    Walshe, Jennifer; Harkin, Damien G

    2014-10-01

    The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.

  10. Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model, LabCyte CORNEA-MODEL.

    Science.gov (United States)

    Katoh, Masakazu; Hamajima, Fumiyasu; Ogasawara, Takahiro; Hata, Ken-ichiro

    2013-12-01

    Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test.

  11. Unilateral partial limbal stem cell deficiency: contralateral versus ipsilateral autologous cultivated limbal epithelial transplantation.

    Science.gov (United States)

    Vazirani, Jayesh; Basu, Sayan; Kenia, Hemal; Ali, Md Hasnat; Kacham, Santhosh; Mariappan, Indumathi; Sangwan, Virender

    2014-03-01

    To report the outcomes of autologous cultivated limbal epithelial transplantation using the healthy part of the affected eye or the fellow eye as a source of limbal stem cells in patients with unilateral, partial limbal stem cell deficiency (LSCD). Retrospective, nonrandomized, interventional case series. setting: L. V. Prasad Eye Institute, Hyderabad, India. study population: Patients with unilateral, partial LSCD who underwent autologous cultivated limbal epithelial transplantation between 2001 and 2011. intervention: The limbal biopsy was taken either from the healthy part of the limbus of the same eye (ipsilateral group) or from the healthy fellow eye (contralateral group). Cells were cultivated using a xeno-free explant culture technique, and cultivated cells were transplanted onto the affected surface. primary outcome measure: Success of cultivated limbal epithelial transplantation, defined as a completely epithelialized, avascular, and clinically stable corneal surface. Seventy eyes of 70 patients were studied. The mean follow up was 17.5 ± 7 months. In 34 eyes the limbal biopsy was taken from the ipsilateral eye and in the remaining 36 eyes from the contralateral eye. Clinical success was achieved in 70.59% of eyes in the ipsilateral group and 75% of eyes in the contralateral group (P = .79). Limbal transplant survival rates at the final follow-up visit were 65.1% ± 0.09% in the ipsilateral group and 53.6% ± 0.12% in the contralateral group (P = .74). Ocular surface restoration in partial LSCD is possible with cell-based therapy. Outcomes are similar irrespective of whether the limbal biopsy is taken from the healthy part of the ipsilateral eye or the contralateral eye. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Evaluation of corneal cell growth on tissue engineering materials as artificial cornea scaffolds

    Directory of Open Access Journals (Sweden)

    Hai-Yan Wang

    2013-12-01

    Full Text Available The keratoprosthesis (KPro; artificial cornea is a special refractive device to replace human cornea by using heterogeneous forming materials for the implantation into the damaged eyes in order to obtain a certain vision. The main problems of artificial cornea are the biocompatibility and stability of the tissue particularly in penetrating keratoplasty. The current studies of tissue-engineered scaffold materials through comprising composites of natural and synthetic biopolymers together have developed a new way to artificial cornea. Although a wide agreement that the long-term stability of these devices would be greatly improved by the presence of cornea cells, modification of keratoprosthesis to support cornea cells remains elusive. Most of the studies on corneal substrate materials and surface modification of composites have tried to improve the growth and biocompatibility of cornea cells which can not only reduce the stimulus of heterogeneous materials, but also more importantly continuous and stable cornea cells can prevent the destruction of collagenase. The necrosis of stroma and spontaneous extrusion of the device, allow for maintenance of a precorneal tear layer, and play the role of ensuring a good optical surface and resisting bacterial infection. As a result, improvement in corneal cells has been the main aim of several recent investigations; some effort has focused on biomaterial for its well biological properties such as promoting the growth of cornea cells. The purpose of this review is to summary the growth status of the corneal cells after the implantation of several artificial corneas.

  13. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Araki Hiromasa

    2007-04-01

    Full Text Available Abstract Background Proteinase-activated receptors (PARs; PAR1–4 that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells. Results Stimulation of PAR with thrombin (1 U/ml or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β. Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial

  14. Establishment of Hertwig’s Epithelial Root Sheath/Epithelial Rests of Malassez Cell Line from Human Periodontium

    OpenAIRE

    Nam, Hyun; Kim, Ji-Hye; Kim, Jae-Won; Seo, Byoung-Moo; Park, Joo-Cheol; Kim, Jung-Wook; Lee, Gene

    2014-01-01

    Human Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare populati...

  15. No junctional communication between epithelial cells in hydra

    DEFF Research Database (Denmark)

    de Laat, S W; Tertoolen, L G; Grimmelikhuijzen, C J

    1980-01-01

    junctions between epithelial cells of hydra. However, until now, there has been no report published on whether these junctions enable the epithelial cells to exchange molecules of small molecular weight, as has been described in other organisms. Therefore we decided to investigate the communicative...... properties of the junctional membranes by electrophysiological methods and by intracellular-dye iontophoresis. We report here that no electrotonic coupling is detectable between epithelial cells of Hydra attenuata in: (1) intact animals, (2) head-regenerating animals, (3) cell re-aggregates, and (4) hydra...

  16. Quantitative assessment of cytosolic Salmonella in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Leigh A Knodler

    Full Text Available Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV. We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1, but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.

  17. Methods Development for the Isolation and Culture of Primary Corneal Endothelial Cells

    Science.gov (United States)

    2017-02-01

    stromal fibroblasts (keratocytes). Additional challenges are encountered in attempts to expand isolated cells in culture while maintaining CEC morphology...maintain normal cobblestone morphology and favorable growth. However, no results were reported in this study for cells that had been expanded in...and development. Prog Mol Biol Transl Sci 2015; 134: 7-23. 2. Joyce NC. Proliferative capacity of the corneal endothelium. Progress in Retinal and

  18. Cytotoxic effects of betaxolol on healthy corneal endothelial cells both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Ying Miao

    2014-02-01

    Full Text Available AIM: To demonstrate the cytotoxic effect of betaxolol and its underlying mechanism on human corneal endothelial cells(HCE cells in vitro and cat corneal endothelial cells(CCE cells in vivo, providing experimental basis for safety anti-glaucoma drug usage in clinic of ophthalmology.METHODS: In vivo and in vitro experiments were conducted to explore whether and how betaxolol participates in corneal endothelial cell injury. The in vitro morphology, growth status, plasma membrane permeability, DNA fragmentation, and ultrastructure of HCE cells treated with 0.021875-0.28g/L betaxolol were examined by light microscope, 3-(4,5-dimethylthiahiazo (-z-y1-3,5-di-phenytetrazoliumromide (MTT assay, acridine orange (AO/ethidium bromide (EB double-fluorescent staining, DNA agarose gel electrophoresis, and transmission electron microscope (TEM. The in vivo density, morphology, and ultrastructure of CCE cells, corneal thickness, and eye pressure of cat eyes treated with 0.28g/L betaxolol were investigated by specular microscopy, applanation tonometer, alizarin red staining, scanning electron microscope (SEM, and TEM.RESULTS: Exposure to betaxolol at doses from 0.0875g/L to 2.8g/L induced morphological and ultrastructural changes of in vitro cultured HCE cells such as cytoplasmic vacuolation, cellular shrinkage, structural disorganization, chromatin condensation, and apoptotic body appearance. Simultaneously, betaxolol elevated plasma membrane permeability and induced DNA fragmentation of these cells in a dose-dependent manner in AO/EB staining. Furthermore, betaxolol at a dose of 2.8g/L also induced decrease of density of CCE cells in vivo, and non-hexagonal and shrunk apoptotic cells were also found in betaxolol-treated cat corneal endothelia.CONCLUSION: Betaxolol has significant cytotoxicity on HCE cells in vitro by inducing apoptosis of these cells, and induced apoptosis of CCE cells in vivo as well. The findings help provide new insight into the apoptosis

  19. Ocular Surface Reconstruction with Cultivated Limbal Epithelial Cells in Limbal Stem Cell Deficiency: One-year Follow-up Results

    Directory of Open Access Journals (Sweden)

    İsmet Durak

    2012-05-01

    Full Text Available Pur po se: To evaluate the 1-year follow-up results of cultivated limbal epithelial cell (CLEC transplantation in unilateral limbal stem cell deficiency (LSCD. Ma te ri al and Met hod: One-year follow-up results of five unilateral LSCD patients who had undergone CLEC transplantation were evaluated. Parameters for this evaluation were: fluorescein staining of ocular surface, corneal vascularization and status of epithelium with slit lamp, and visual acuity. 1.5-mm limbal biopsy was performed from the superior limbus of the healthy eyes, broke into two equal pieces, expanded on human amniotic membrane (hAM and inserts for 14 days until getting 20 mm in size. CLECs on hAMs were used directly, and cells on inserts were usedafter detachment procedure. The symblepharon and pannus tissues were removed, superficial keratectomy was performed. CLEC on hAMs were transplanted with the epithelial side up onto the bare corneal stroma, sutured to the conjunctiva with 10-0 nylon sutures. Free CLEC layer from insert was placed on hAM as a second layer, additional hAM was used as a protective layer all over other tissues. Re sults: Median age was 44.4 years (14-71. The etiology was chemical burn in all patients. Median duration of symptoms was 10 years (2-18, median follow-up period was 12.6 (12-12.5 months. Preoperative best corrected visual acuities (BCVA were light perception in three patients, counting fingers at 50 cm in one patient and 3/10 in one patient. Visions were improved in all patients. Postoperative BCVA 12 months after the surgery were between counting fingers at 3 meters to 6/10. There was a temporary hemorrhage between the two layers of hAMs in one patient at the early postoperative period. Peripheral corneal vascularization has occurred in three patients, in patient corneal vascularization has reached to the paracentral area. Dis cus si on: CLEC transplantation is an efficient treatment option for unilateral LSCD in mid-long term. (Turk J

  20. Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

    NARCIS (Netherlands)

    P. Aparicio-Domingo (Patricia); M. Romera-Hernandez (Monica); J.J. Karrich (Julien J.); F.H.J. Cornelissen (Ferry); N. Papazian (Natalie); D.J. Lindenbergh-Kortleve (Dicky); J.A. Butler (James A.); L. Boon (Louis); M. Coles (Mark); J.N. Samsom (Janneke); T. Cupedo (Tom)

    2015-01-01

    textabstractDisruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence

  1. Sodium selectivity of Reissner's membrane epithelial cells

    Directory of Open Access Journals (Sweden)

    Kim Kyunghee X

    2011-02-01

    Full Text Available Abstract Background Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC, which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196, RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3. By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath. Conclusions These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala

  2. Ion transport in epithelial spheroids derived from human airway cells

    DEFF Research Database (Denmark)

    Pedersen, P S; Frederiksen, O; Holstein-Rathlou, N H

    1999-01-01

    In the present study, we describe a novel three-dimensional airway epithelial explant preparation and demonstrate its use for ion transport studies by electrophysiological technique. Suspension cultures of sheets of epithelial cells released by protease treatment from cystic fibrosis (CF) and non...

  3. VISUAL PERCEPTION BASED AUTOMATIC RECOGNITION OF CELL MOSAICS IN HUMAN CORNEAL ENDOTHELIUMMICROSCOPY IMAGES

    Directory of Open Access Journals (Sweden)

    Yann Gavet

    2011-05-01

    Full Text Available The human corneal endothelium can be observed with two types of microscopes: classical optical microscope for ex-vivo imaging, and specular optical microscope for in-vivo imaging. The quality of the cornea is correlated to the endothelial cell density and morphometry. Automatic methods to analyze the human corneal endothelium images are still not totally efficient. Image analysis methods that focus only on cell contours do not give good results in presence of noise and of bad conditions of acquisition. More elaborated methods introduce regional informations in order to performthe cell contours completion, thus implementing the duality contour-region. Their good performance can be explained by their connections with several basic principles of human visual perception (Gestalt Theory and Marr's computational theory.

  4. Andrographolide suppresses epithelial mesenchymal transition by inhibition of MAPK signalling pathway in lens epithelial cells

    Indian Academy of Sciences (India)

    Forum Kayastha; Kaid Johar; Devarshi Gajjar; Anshul Arora; Hardik Madhu; Darshini Ganatra; Abhay Vasavada

    2015-06-01

    Epithelial mesenchymal transition (EMT) of lens epithelial cells (LECs) may contribute to the development of posterior capsular opacification (PCO), which leads to visual impairment. Andrographolide has been shown to have therapeutic potential against various cancers. However, its effect on human LECs is still unknown. The purpose of this study is to evaluate the effect of andrographolide on EMT induced by growth factors in the fetal human lens epithelial cell line (FHL 124). Initially the LECs were treated with growth factors (TGF-2 and bFGF) to induce EMT. Subsequently these EMT-induced cells were treated with andrographolide at 100 and 500 nM concentrations for 24 h. Our results showed that FHL 124 cells treated with growth factors had a significant decrease in protein and m-RNA levels of epithelial markers pax6 and E-Cadherin. After administering andrographolide, these levels significantly increased. It was noticed that EMT markers -SMA, fibronectin and collagen IV significantly decreased after treatment with andrographolide when compared to the other group. Treatment with andrographolide significantly inhibited phosphorylation of ERK and JNK. Cell cycle analysis showed that andrographolide did not arrest cells at G0/G1 or G2/M at tested concentrations. Our findings suggest that andrographolide helps sustain epithelial characteristics by modulating EMT markers and inhibiting the mitogen-activated protein kinase (MAPK) signalling pathway in LECs. Hence it can prove to be useful in curbing EMT-mediated PCO.

  5. Cells from the adult corneal stroma can be reprogrammed to a neuron-like cell using exogenous growth factors

    Energy Technology Data Exchange (ETDEWEB)

    Greene, Carol Ann, E-mail: carol.greene@auckland.ac.nz; Chang, Chuan-Yuan; Fraser, Cameron J.; Nelidova, Dasha E.; Chen, Jing A.; Lim, Angela; Brebner, Alex; McGhee, Jennifer; Sherwin, Trevor; Green, Colin R.

    2014-03-10

    Cells thought to be stem cells isolated from the cornea of the eye have been shown to exhibit neurogenic potential. We set out to uncover the identity and location of these cells within the cornea and to elucidate their neuronal protein and gene expression profile during the process of switching to a neuron-like cell. Here we report that every cell of the adult human and rat corneal stroma is capable of differentiating into a neuron-like cell when treated with neurogenic differentiation specifying growth factors. Furthermore, the expression of genes regulating neurogenesis and mature neuronal structure and function was increased. The switch from a corneal stromal cell to a neuron-like cell was also shown to occur in vivo in intact corneas of living rats. Our results clearly indicate that lineage specifying growth factors can affect changes in the protein and gene expression profiles of adult cells, suggesting that possibly many adult cell populations can be made to switch into another type of mature cell by simply modifying the growth factor environment. - Highlights: • Adult corneal stromal cells can differentiated into neuron-like cells. • Neuronal specification of the adult stromal cell population is stochastic. • Neuronal specification in an adult cell population can be brought about by growth factors.

  6. Endoplasmic reticulum protein 29 regulates epithelial cell integrity during the mesenchymal-epithelial transition in breast cancer cells.

    Science.gov (United States)

    Bambang, I F; Lee, Y K; Richardson, D R; Zhang, D

    2013-03-07

    The epithelial-mesenchymal transition (EMT) correlates with disruption of cell-cell adhesion, loss of cell polarity and development of epithelial cell malignancy. Identifying novel molecules that inhibit EMT has profound potential for developing mechanism-based therapeutics. We previously demonstrated that the endoplasmic reticulum protein 29 (ERp29) is a novel factor that can drive mesenchymal-epithelial transition (MET) and induce cell growth arrest in MDA-MB-231 cells. Here, we show that ERp29 is an important molecule in establishing epithelial cell integrity during the MET. We demonstrate that ERp29 regulates MET in a cell context-dependent manner. ERp29 overexpression induced a complete MET in mesenchymal MDA-MB-231 cells through downregulating the expression of transcriptional repressors (for example, Slug, Snai1, ZEB2 and Twist) of E-cadherin. In contrast, overexpression of ERp29 induces incomplete MET in basal-like BT549 cells in which the expression of EMT-related markers (for example, vimentin; cytokeratin 19 (CK19) and E-cadherin) and the transcriptional repressors of E-cadherin were not altered. However, ERp29 overexpression in both cell-types resulted in loss of filamentous stress fibers, formation of cortical actin and restoration of an epithelial phenotype. Mechanistic studies revealed that overexpression of ERp29 in both cell-types upregulated the expression of TJ proteins (zonula-occludens-1 (ZO-1) and occludin) and the core apical-basal polarity proteins (Par3 and Scribble) at the membrane to enhance cell-cell contact and cell polarization. Knockdown of ERp29 in the epithelial MCF-7 cells decreased the expression of these proteins, leading to the disruption of cell-cell adhesion. Taken together, ERp29 is a novel molecule that regulates MET and epithelial cell integrity in breast cancer cells.

  7. Mitosis orientation in prostate epithelial cells changed by endocrine effect

    Institute of Scientific and Technical Information of China (English)

    Xiang-yun LIU; Dong-mei Li; Xiao-fang ZHANG; Jian-hui WU; Zu-yue SUN

    2008-01-01

    Aim: The aim of the present study was to investigate the effect of androgen and estrogen on mitosis orientation in the prostate epithelial cells of male rats. Methods: Castrated rats were treated with a single injection of testosterone propionate (TP) or benzogynestry (E2). There were 8 rats in the control group and TP-treated or E2-treated group. Prostate, liver, a specimen of skin, and a segment of the jejunum and colon were removed after the corresponding treatment. The results were observed through immunohistochemistry and iron hematoxylin-eosin staining.Results: All mitoses found in the prostate epithelial cells of castrated rats with TP were oriented parallel to the basement membrane; however, mitoses found in the prostate epithelial cells of castrated rats in E2 and the control group were oriented perpendicular to the basement membrane. TP treatment resulted in marked changes in mitosis orientation in the prostate epithelial cells. Bromodeoxyuridine-labeled positive cells could be seen throughout the stroma and prostate epithelial cells with an injection of TP; however, the positive cells could only be seen in the stroma of prostate with an injection of E2, and the positive cells could hardly be seen in the control group. Conclusion: We found a novel effect of TP in the prostate as a marked change of mitosis orientation in prostate epithelial cells.

  8. Cell cycle regulation by glucosamine in human pulmonary epithelial cells.

    Science.gov (United States)

    Chuang, Kun-Han; Lu, Chih-Shen; Kou, Yu Ru; Wu, Yuh-Lin

    2013-04-01

    Airway epithelial cells play an important role against intruding pathogens. Glucosamine, a commonly used supplemental compound, has recently begun to be regarded as a potential anti-inflammatory molecule. This study aimed to uncover how glucosamine impacts on cellular proliferation in human alveolar epithelial cells (A549) and bronchial epithelial cells (HBECs). With trypan blue-exclusion assay, we observed that glucosamine (10, 20, 50 mM) caused a decrease in cell number at 24 and 48 h; with a flow cytometric analysis, we also noted an enhanced cell accumulation within the G(0)/G(1) phase at 24 h and induction of late apoptosis at 24 and 48 h by glucosamine (10, 20, 50 mM) in A549 cells and HBECs. Examination of phosphorylation in retinoblastoma (Rb) protein, we found an inhibitory effect by glucosamine at 20 and 50 mM. Glucosamine at 50 mM was demonstrated to elevate both the mRNA and protein expression of p53 and heme oxygenase-1 (HO-1), but also caused a reduction in p21 protein expression. In addition, glucosamine attenuated p21 protein stability via the proteasomal proteolytic pathway, as well as inducing p21 nuclear accumulation. Altogether, our results suggest that a high dose of glucosamine may inhibit cell proliferation through apoptosis and disturb cell cycle progression with a halt at G(0)/G(1) phase, and that this occurs, at least in part, by a reduction in Rb phosphorylation together with modulation of p21, p53 and HO-1 expression, and nuclear p21 accumulation.

  9. Simvastatin Attenuates TGF-β1-Induced Epithelial-Mesenchymal Transition in Human Alveolar Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Tuo Yang

    2013-06-01

    Full Text Available Background: Transforming growth factor-β1 (TGF-β1-induced epithelial-mesenchymal transition (EMT of alveolar epithelial cells (AEC may contribute to idiopathic pulmonary fibrosis (IPF. TGF-β1-induced EMT in A549 cells (a human AEC cell line resulted in the adoption of mesenchymal responses that were predominantly mediated via the TGF-β1-Smad2/3 signaling pathway. Simvastatin (Sim, a 3-hydroxy-3-methylglutaryl CoA (HMG-CoA reductase inhibitor, has been previously reported to inhibit EMT in human proximal tubular epithelial cells and porcine lens epithelial cells and to suppress Smad2/3 phosphorylation in animal models. However, whether Sim can attenuate TGF-β1-induced EMT in A549 cells and its underlying mechanisms remains unknown. Methods: Cells were incubated with TGF-β1 in the presence or absence of Sim. The epithelial marker E-cadherin (E-Cad and the mesenchymal markers, α-smooth muscle actin (α-SMA, vimentin (Vi and fibronectin (FN, were detected using western blotting analyses and immunofluorescence. Phosphorylated Smad2 and Smad3 levels and connective tissue growth factor (CTGF were analyzed using western blotting. In addition, a cell migration assay was performed. Moreover, the levels of matrix metalloproteinase (MMP-2 and -9 in the culture medium were examined using ELISA. Results: Sim significantly attenuated the TGF-β1-induced decrease in E-Cad levels and elevated the levels of α-SMA, Vi and FN via the suppression of Smad2 and Smad3 phosphorylation. Furthermore, Sim inhibited the mesenchymal-like responses in A549 cells, including cell migration, CTGF expression and secretion of MMP-2 and -9. However, Sim failed to reverse the cell morphologial changes induced by TGF-β1 in A549 cells. Conclusion: Sim attenuated TGF-β1-induced EMT in A549 cells and might be a promising therapeutic agent for treating IPF.

  10. Cell volume regulation in epithelial physiology and cancer

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Hoffmann, Else Kay; Novak, Ivana

    2013-01-01

    The physiological function of epithelia is transport of ions, nutrients, and fluid either in secretory or absorptive direction. All of these processes are closely related to cell volume changes, which are thus an integrated part of epithelial function. Transepithelial transport and cell volume...... expression of ion transporters and channels is now recognized as one of the hallmarks of cancer, it is timely to consider this especially for epithelia. Epithelial cells are highly proliferative and epithelial cancers, carcinomas, account for about 90% of all cancers. In this review we will focus on ion...... transporters and channels with key physiological functions in epithelia and known roles in the development of cancer in these tissues. Their roles in cell survival, cell cycle progression, and development of drug resistance in epithelial cancers will be discussed....

  11. Inhibition of TGF-β signaling enables human corneal endothelial cell expansion in vitro for use in regenerative medicine.

    Directory of Open Access Journals (Sweden)

    Naoki Okumura

    Full Text Available Corneal endothelial dysfunctions occurring in patients with Fuchs' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na(+/K(+-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions.

  12. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Science.gov (United States)

    Chou, Ming-Li; Burnouf, Thierry; Wang, Tsung-Jen

    2014-01-01

    Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory

  13. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Directory of Open Access Journals (Sweden)

    Ming-Li Chou

    Full Text Available Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices

  14. Rac promotes epithelial cell rearrangement during tracheal tubulogenesis in Drosophila.

    Science.gov (United States)

    Chihara, Takahiro; Kato, Kagayaki; Taniguchi, Misako; Ng, Julian; Hayashi, Shigeo

    2003-04-01

    Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. We used the tracheal system of Drosophila to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. We found that a reduced Rac activity led to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibited dynamic epithelial cell rearrangement. Hyperactivation of Rac, on the other hand, inhibited assembly of newly synthesized E-Cadherin into cell junctions and caused loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules.

  15. Differential expression of the Slc4 bicarbonate transporter family in murine corneal endothelium and cell culture.

    Science.gov (United States)

    Shei, William; Liu, Jun; Htoon, Hla M; Aung, Tin; Vithana, Eranga N

    2013-01-01

    To characterize the relative expression levels of all the solute carrier 4 (Slc4) transporter family members (Slc4a1-Slc4a11) in murine corneal endothelium using real-time quantitative (qPCR), to identify further important members besides Slc4a11 and Slc4a4, and to explore how close to the baseline levels the gene expressions remain after cells have been subjected to expansion and culture. Descemet's membrane-endothelial layers of 8-10-week-old C57BL6 mice were stripped from corneas and used for both primary cell culture and direct RNA extraction. Total RNA (from uncultured cells as well as cultured cells at passages 2 and 7) was reverse transcribed, and the cDNA was used for real time qPCR using specific primers for all the Slc4 family members. The geNorm method was applied to determine the most stable housekeeping genes and normalization factor, which was calculated from multiple housekeeping genes for more accurate and robust quantification. qPCR analyses revealed that all Slc4 bicarbonate transporter family members were expressed in mouse corneal endothelium. Slc4a11 showed the highest expression, which was approximately three times higher than that of Slc4a4 (3.4±0.3; p=0.004). All Slc4 genes were also expressed in cultured cells, and interestingly, the expression of Slc4a11 in cultured cells was significantly reduced by approximately 20-fold (0.05±0.001; p=0.000001) in early passage and by approximately sevenfold (0.14±0.002; p=0.000002) in late passage cells. Given the known involvement of SLC4A4 and SLC4A11 in corneal dystrophies, we speculate that the other two highly expressed genes in the uncultured corneal endothelium, SLC4A2 and SLC4A7, are worthy of being considered as potential candidate genes for corneal endothelial diseases. Moreover, as cell culture can affect expression levels of Slc4 genes, caution and careful design of experiments are necessary when undertaking studies of Slc4-mediated ion transport in cultured cells.

  16. Corneal Laceration

    Medline Plus

    Full Text Available ... Laceration? Corneal Laceration Diagnosis Corneal Laceration Treatment What Is Corneal Laceration? Written By: Daniel Porter Reviewed By: ... A Harrison MD Sep. 01, 2016 The cornea is the clear front window of the eye . A ...

  17. Potential uses of milk epithelial cells: a review.

    Science.gov (United States)

    Boutinaud, Marion; Jammes, Hélène

    2002-01-01

    Secretions collected from the mammary gland of different species contain heterogeneous populations of cells including lymphocytes, neutrophils, macrophages and epithelial cells in different species. Several factors influence the somatic cell count in milk and the distribution of cell types, such as species, infection status, physiological status and management practices. The epithelial cells are shed into milk during the lactation process. Most of them are viable and exhibit the characteristics of fully differentiated alveolar cells. Primary cultures of epithelial cells from colostrum and milk of humans, baboons, cows and goats together with established cell lines from human and goat milk, provide a good model for the study of lactogenesis, immunity transmission, cancer research and infection by viruses. The RNA extracted from milk cells have been shown to be representative of gene expression in the mammary gland and thus provide a source of material for molecular studies of gene expression and environmental interactions.

  18. Primary Corneal Squamous Cell Carcinoma in a Dog: Clinical and Histopathological Evaluation

    OpenAIRE

    Giovanni Barsotti; Lorenzo Ressel; Riccardo Finotello; Veronica Marchetti; Francesca Millanta

    2012-01-01

    An 8-year-old male pug with a 12-month history of a progressive nonpainful mass on the left cornea was evaluated. Ocular examination showed a severe bilateral keratoconjunctivitis sicca, pigmentary keratitis, and an exophytic irregular pink mass occupying approximately 75% of the total corneal surface of the left eye. A squamous cell carcinoma (SCC) was suspected on cytology, and clinical investigations showed no evidence of metastases. A transpalpebral enucleation was therefore performed, an...

  19. Mitochondrial and Morphologic Alterations in Native Human Corneal Endothelial Cells Associated With Diabetes Mellitus.

    Science.gov (United States)

    Aldrich, Benjamin T; Schlötzer-Schrehardt, Ursula; Skeie, Jessica M; Burckart, Kimberlee A; Schmidt, Gregory A; Reed, Cynthia R; Zimmerman, M Bridget; Kruse, Friedrich E; Greiner, Mark A

    2017-04-01

    To characterize changes in the energy-producing metabolic activity and morphologic ultrastructure of corneal endothelial cells associated with diabetes mellitus. Transplant suitable corneoscleral tissue was obtained from donors aged 50 to 75 years. We assayed 3-mm punches of endothelium-Descemet membrane for mitochondrial respiration and glycolysis activity using extracellular flux analysis of oxygen and pH, respectively. Transmission electron microscopy was used to assess qualitative and quantitative ultrastructural changes in corneal endothelial cells and associated Descemet membrane. For purposes of analysis, samples were divided into four groups based on a medical history of diabetes regardless of type: (1) nondiabetic, (2) noninsulin-dependent diabetic, (3) insulin-dependent diabetic, and (4) insulin-dependent diabetic with specified complications due to diabetes (advanced diabetic). In total, 229 corneas from 159 donors were analyzed. Insulin-dependent diabetic samples with complications due to diabetes displayed the lowest spare respiratory values compared to all other groups (P ≤ 0.002). The remaining mitochondrial respiration and glycolysis metrics did not differ significantly among groups. Compared to nondiabetic controls, the endothelium from advanced diabetic samples had alterations in mitochondrial morphology, pronounced Golgi bodies associated with abundant vesicles, accumulation of lysosomal bodies/autophagosomes, and focal production of abnormal long-spacing collagen. Extracellular flux analysis suggests that corneal endothelial cells of donors with advanced diabetes have impaired mitochondrial function. Metabolic findings are supported by observed differences in mitochondrial morphology of advanced diabetic samples but not controls. Additional studies are needed to determine the precise mechanism(s) by which mitochondria become impaired in diabetic corneal endothelial cells.

  20. Deletion of JAM-A causes morphological defects in the corneal epithelium.

    Science.gov (United States)

    Kang, Liang I; Wang, Yan; Suckow, Arthur T; Czymmek, Kirk J; Cooke, Vesselina G; Naik, Ulhas P; Duncan, Melinda K

    2007-01-01

    Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.

  1. In Vitro transformation of LW13 Rat liver epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    SHICAN; KARLFETNANSKY; 等

    1992-01-01

    A rat liver epithelial cell line designated LW 13 was established using a sequential sedimentation method.The cell line retained many normal proerties of liver epithelial cells and showed some structural and functional features resembling those of liver parenchymal cells,LW13 cells became malignant after the intrduction of exogenous transforming EJ Ha ras gene,Tumors produced by inoculation of the transformed cells into baby rats contained areas of poorly differentialted hepatocellular carcinoma,In situ hybridization analysis confirmed the random rather than specific integration of exogenous ras gene into host chromosomes.Furthermore,an at least tenfold increase in the expression of the endogenous c mys gene was detected among transformed cell lines,suggesting the involvement of the c myc proto oncogene in the in vitro transformation of rat liver epithelial cells by EJ Ha ras oncogene.

  2. Pirfenidone Nanoparticles Improve Corneal Wound Healing and Prevent Scarring Following Alkali Burn

    OpenAIRE

    Sushovan Chowdhury; Rajdeep Guha; Ruchit Trivedi; Kompella, Uday B.; Aditya Konar; Sarbani Hazra

    2013-01-01

    PURPOSE: To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn. METHODS: Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA) synthesis by TGFβ induced primary corneal fibroblast cells was evaluated by immunoblotting and immunocytochemistry. Pirfenidone loaded poly (lactide-co-glycolide) (PLGA) nanoparticles were prepared, characterized and their cellular entry was examined in primary cor...

  3. Self-organized centripetal movement of corneal epithelium in the absence of external cues

    Science.gov (United States)

    Lobo, Erwin P.; Delic, Naomi C.; Richardson, Alex; Raviraj, Vanisri; Halliday, Gary M.; Di Girolamo, Nick; Myerscough, Mary R.; Lyons, J. Guy

    2016-01-01

    Maintaining the structure of the cornea is essential for high-quality vision. In adult mammals, corneal epithelial cells emanate from stem cells in the limbus, driven by an unknown mechanism towards the centre of the cornea as cohesive clonal groups. Here we use complementary mathematical and biological models to show that corneal epithelial cells can self-organize into a cohesive, centripetal growth pattern in the absence of external physiological cues. Three conditions are required: a circumferential location of stem cells, a limited number of cell divisions and mobility in response to population pressure. We have used these complementary models to provide explanations for the increased rate of centripetal migration caused by wounding and the potential for stem cell leakage to account for stable transplants derived from central corneal tissue, despite the predominantly limbal location of stem cells. PMID:27499113

  4. Self-organized centripetal movement of corneal epithelium in the absence of external cues

    Science.gov (United States)

    Lobo, Erwin P.; Delic, Naomi C.; Richardson, Alex; Raviraj, Vanisri; Halliday, Gary M.; di Girolamo, Nick; Myerscough, Mary R.; Lyons, J. Guy

    2016-08-01

    Maintaining the structure of the cornea is essential for high-quality vision. In adult mammals, corneal epithelial cells emanate from stem cells in the limbus, driven by an unknown mechanism towards the centre of the cornea as cohesive clonal groups. Here we use complementary mathematical and biological models to show that corneal epithelial cells can self-organize into a cohesive, centripetal growth pattern in the absence of external physiological cues. Three conditions are required: a circumferential location of stem cells, a limited number of cell divisions and mobility in response to population pressure. We have used these complementary models to provide explanations for the increased rate of centripetal migration caused by wounding and the potential for stem cell leakage to account for stable transplants derived from central corneal tissue, despite the predominantly limbal location of stem cells.

  5. Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirai, H.; Lochter, A.; Galosy, S.; Koshida, S.; Niwa, S.; Bissell, M.J.

    1997-10-13

    Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

  6. Connective Tissue Growth Factor Expression in Human Bronchial Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Amrita DOSANJH

    2006-01-01

    Connective tissue growth factor (CTGF) is a cysteine-rich protein that promotes extracellular matrix deposition. CTGF is selectively induced by transforming growth factor β and des-Arg kallidin in lung fibroblasts and increases steady-state mRNA levels of α type I collagen, 5α-integrin and fibronectin in fibroblasts. Bronchial epithelial cells have been proposed to functionally interact with lung fibroblasts. We therefore investigated if bronchial epithelial cells are able to synthesize CTGF. Human bronchial epithelial cells were grown to subconfluence in standard growth media. Proliferating cells grown in small airway growth media were harvested following starvation for up to 24 h. Expression of CTGF transcripts was measured by PCR. Immunocytochemistry was also completed using a commercially available antibody.The cells expressed readily detectable CTGF transcripts. Starvation of these cells resulted in a quantitative decline of CTGF transcripts. Direct sequencing of the PCR product identified human CTGF. Immunocytochemistry confirmed intracellular CTGF in the cells and none in negative control cells. We conclude that bronchial epithelial cells could be a novel source of CTGF. Bronchial epithelial cell-derived CTGF could thus directly influence the deposition of collagen in certain fibrotic lung diseases.

  7. Corneal surface reconstruction - a short review

    Directory of Open Access Journals (Sweden)

    Madhavan H N

    2009-01-01

    Full Text Available Cornea is the clear, dome-shaped surface that covers the front of the eye and when damage due to burns or injury and several other diseases, stem cells residing in its rim called "limbus" are stimulated to multiply to support growth of new epithelial cells over its surface. If this ready source of stem cells is damaged or destroyed the natural repair is not possible and such a condition is known as corneal limbal stem cell deficiency (CLSCD disease. Stem cell transplant helps such persons to regenerate the corneal surface. Human corneal limbal stem cell transplantation is at present an established procedure with reasonable good clinical outcome particularly when autologous limbal epithelial tissue from a fellow unaffected eye is used. 1, 2 A major concern related to the autograft is the possibility of CLSCD at the donor site, 3 techniques that allowed the expansion of a small limbal biopsy in the laboratory using cell cultures that could be then transplanted to the affected eye have been developed ,4, 5 Human amniotic membrane (HAM is used as a scaffold for both culturing the human limbal epithelial cells and for ocular surface reconstruction with the cultured limbal epithelial cells. 4-7 However, researchers have used alternative scaffolds like collagen 8, fibrin gel 9 and cross-linked gel of fibronectin and fibrin. 10 All these are biological materials and also need for animal 3T3 feeder layer for stem cell cultures. The properties of HAM are unique including antiadhesive effects, bacteriostatic effects, wound protection, pain reduction, and improvement of epithelialization and characteristically lacking imunogenicity. The use of amniotic membrane transplantation (AMT to treat ocular surface abnormalities was first reported by Graziella Pellegrini, chief of stem cell laboratory at Giovanni Paolo Hospital in Venice, Italy, who was the first to demonstrate the limbal stem cell transplant in 1997. Amniotic membrane has been successfully used in

  8. Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition.

    Science.gov (United States)

    Hong, Deli; Messier, Terri L; Tye, Coralee E; Dobson, Jason R; Fritz, Andrew J; Sikora, Kenneth R; Browne, Gillian; Stein, Janet L; Lian, Jane B; Stein, Gary S

    2017-03-14

    Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFβ signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFβ and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression.

  9. Attachment of epithelial cells and fibroblasts to ceramic materials.

    Science.gov (United States)

    Niederauer, G G; McGee, T D; Keller, J C; Zaharias, R S

    1994-04-01

    This study examined in vitro gingival epithelial and fibroblast cell attachment to ceramic materials made of tricalcium phosphate and/or magnesium aluminate spinel. The composite made of tricalcium phosphate and spinel is called 'osteoceramic'. These ceramics had various compositions and surface structures, which were initially characterized. Cell attachment assays were performed using both cell types to compare cellular response to the ceramic materials. Specimens were also prepared for scanning electron microscopy to investigate cellular morphology. The highest levels of cell attachment for gingival epithelial cells were observed on the rough osteoceramic surface, whereas gingival fibroblasts attached least to the rough osteoceramic surface.

  10. Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels.

    Directory of Open Access Journals (Sweden)

    Philipp Steven

    Full Text Available BACKGROUND: The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. METHODOLOGY/PRINCIPAL FINDINGS: We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM. Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. CONCLUSIONS: To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible

  11. Probiotics promote endocytic allergen degradation in gut epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Song, Chun-Hua [Department of Epidemiology and Biostatistics, College of Public Health, Zhengzhou University, Zhengzhou (China); Liu, Zhi-Qiang [Department of Gastroenterology, The Second Hospital, Zhengzhou University, Zhengzhou (China); Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON (Canada); Huang, Shelly [Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON (Canada); Zheng, Peng-Yuan, E-mail: medp7123@126.com [Department of Gastroenterology, The Second Hospital, Zhengzhou University, Zhengzhou (China); Yang, Ping-Chang, E-mail: yangp@mcmaster.ca [Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON (Canada)

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barrier function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.

  12. Impact of temporary hyperthermia on corneal endothelial cell survival during organ culture preservation.

    Science.gov (United States)

    Schroeter, Jan; Ruggeri, Alfredo; Thieme, Hagen; Meltendorf, Christian

    2015-05-01

    To evaluate temporary exposure to hyperthermia for its impact on endothelial cell density of porcine corneas in organ culture medium containing dextran with regards to possible negative influences of high temperatures during the storage and transport of corneal grafts. Four groups of central discs (diameter 8 mm) from the corneas of both eyes in 40 pigs were first organ-cultured (MEM with 6% dextran 500) for 24 h at 32°C. Ten corneas were then exposed to 40°C in group 1, to 42°C in group 2, to 44°C in group 3, and to 50°C in group 4 for 12 h each. The paired corneal discs for all groups were not treated, stored at 32°C and served as controls. After further organ culture of all corneas for 48 h at 32°C to allow regenerative processes, corneal endothelium was stained with Alizarin Red S and examined by light microscopy. The endothelial cell densities were determined on three central images using a system for the automatic estimation of morphometric parameters of corneal endothelium. Exposure for 12 h to 40°C as well as to 42°C induced no endothelial cell loss. Statistical analysis showed no significant difference of the endothelial cell density between corneas exposed to 40°C and 42°C and the control corneas (40°C treatment: 4736 ± 426 cells/mm(2) and control: 4762 ± 344 cells/mm(2), p = 0.74; 42°C treatment: 4240 ± 363 cells/mm(2) and control: 4176 ± 448 cells/mm(2), p = 0.40). Exposure to 44°C and 50°C lead to total necrosis of the endothelial cell layer. Exposure of organ cultured porcine corneas in dextran containing medium up to 42°C for 12 h does not compromise the endothelial cell density in a clinically relevant manner. Temperatures above 42°C, as it might be the case during transports from the cornea bank to the ophthalmic surgeon, must be strictly avoided as they damage the endothelial cell layer.

  13. Characterisation of cell adhesion in airway epithelial cell types using electric cell-substrate impedance sensing

    NARCIS (Netherlands)

    Heijink, I H; Brandenburg, S M; Noordhoek, J A; Postma, D S; Slebos, D-J; van Oosterhout, A J M

    Research on epithelial cell lines and primary epithelium is required to dissect the mechanisms underlying the structural abnormalities in airway epithelium observed for respiratory diseases, including asthma and chronic obstructive pulmonary disease. The novel electric cell-substrate impedance

  14. Lingual Epithelial Stem Cells and Organoid Culture of Them.

    Science.gov (United States)

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-28

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  15. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha

    2016-01-01

    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  16. Starved epithelial cells uptake extracellular matrix for survival

    Science.gov (United States)

    Muranen, Taru; Iwanicki, Marcin P.; Curry, Natasha L.; Hwang, Julie; DuBois, Cory D.; Coloff, Jonathan L.; Hitchcock, Daniel S.; Clish, Clary B.; Brugge, Joan S.; Kalaany, Nada Y.

    2017-01-01

    Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize β4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell β4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition. PMID:28071763

  17. Cell volume regulation in epithelial physiology and cancer

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Hoffmann, Else Kay; Novak, Ivana

    2013-01-01

    The physiological function of epithelia is transport of ions, nutrients, and fluid either in secretory or absorptive direction. All of these processes are closely related to cell volume changes, which are thus an integrated part of epithelial function. Transepithelial transport and cell volume...... transporters and channels with key physiological functions in epithelia and known roles in the development of cancer in these tissues. Their roles in cell survival, cell cycle progression, and development of drug resistance in epithelial cancers will be discussed....... such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered...

  18. Role of p53 in Mammary Epithelial Cell Senescence

    Science.gov (United States)

    2009-05-01

    culture of normal human breast epithelial cells. Methods Cell Biol 1980, 21B:107-135. 23. Easty GC, Easty DM, Monaghan P, Ormerod MG, Neville AM...27, 2006 Monitoring Editor: John Cleveland Polycomb group (PcG) protein Bmi-1 is an important regulator of cell proliferation. It regulates cellular

  19. An Ultra-thin Amniotic Membrane as Carrier in Corneal Epithelium Tissue-Engineering.

    Science.gov (United States)

    Zhang, Liying; Zou, Dulei; Li, Sanming; Wang, Junqi; Qu, Yangluowa; Ou, Shangkun; Jia, Changkai; Li, Juan; He, Hui; Liu, Tingting; Yang, Jie; Chen, Yongxiong; Liu, Zuguo; Li, Wei

    2016-02-15

    Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 μm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.

  20. Gr-1intCD11b+ myeloid-derived suppressor cells accumulate in corneal allograft and improve corneal allograft survival.

    Science.gov (United States)

    Choi, Wungrak; Ji, Yong Woo; Ham, Hwa-Yong; Yeo, Areum; Noh, Hyemi; Jin, Su-Eon; Song, Jong Suk; Kim, Hyeon Chang; Kim, Eung Kwon; Lee, Hyung Keun

    2016-12-01

    We identified the characteristics of myeloid-derived suppressor cells (MDSCs) and investigated their mechanism of induction and their functional role in allograft rejection using a murine corneal allograft model. In mice, MDSCs coexpress CD11b and myeloid differentiation antigen Gr-1. Gr-1(+)CD11b(+) cells infiltrated allografted corneas between 4 d and 4 wk after surgery; however, the frequencies of Gr-1(+)CD11b(+) cells were not different between accepted and rejected allografts or in peripheral blood or BM. Of interest, Gr-1(int)CD11b(+) cells, but not Gr-1(hi)CD11b(+) cells, infiltrated the accepted graft early after surgery and expressed high levels of immunosuppressive cytokines, including IL-10, TGF-β, and TNF-related apoptosis-inducing ligand. This population remained until 4 wk after surgery. In vitro, only high dose (>100 ng/ml) of IFN-γ plus GM-CSF could induce immunosuppressive cytokine expression in Gr-1(int)CD11b(+) cells. Furthermore, adoptive transfer of Gr-1(int)CD11b(+) cells reduced T cell infiltration, which improved graft survival. In conclusion, high-dose IFN-γ in allograft areas is essential for development of Gr-1(int)CD11b(+) MDSCs in corneal allografts, and subtle environmental changes in the early period of the allograft can result in a large difference in graft survival.

  1. Epithelial cell division in the Xenopus laevis embryo during gastrulation.

    Science.gov (United States)

    Hatte, Guillaume; Tramier, Marc; Prigent, Claude; Tassan, Jean-Pierre

    2014-01-01

    How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

  2. Change in Cell Shape Is Required for Matrix Metalloproteinase-Induced Epithelial-Mesenchymal Transition of Mammary Epithelial Cells

    Science.gov (United States)

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2010-01-01

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a “cuboidal” epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-β-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents. PMID:18506791

  3. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  4. Biomaterial surface proteomic signature determines interaction with epithelial cells.

    Science.gov (United States)

    Abdallah, Mohamed-Nur; Tran, Simon D; Abughanam, Ghada; Laurenti, Marco; Zuanazzi, David; Mezour, Mohamed A; Xiao, Yizhi; Cerruti, Marta; Siqueira, Walter L; Tamimi, Faleh

    2017-03-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices.

  5. Bradykinin-mediated cell proliferation depends on transactivation of EGF receptor in corneal fibroblasts.

    Science.gov (United States)

    Cheng, Ching-Yi; Tseng, Hui-Ching; Yang, Chuen-Mao

    2012-04-01

    In previous studies, bradykinin (BK) has been shown to induce cell proliferation through BK B2 receptor (B2R) via p42/p44 MAPK in Statens Seruminstitut Rabbit Corneal Cells (SIRCs). In addition to this pathway, EGFR transactivation pathway has been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we further investigate whether these transactivation mechanisms participating in BK-induced cell proliferation in SIRCs. Using an immunofluorescence staining and RT-PCR, we initially characterize that SIRCs were corneal fibroblasts and predominantly expressed B2R by BK. Inhibition of p42/p44 MAPK by the inhibitors of Src, EGFR, and Akt or transfection with respective siRNAs prevents BK-induced DNA synthesis in SIRCs. The mechanisms underlying these responses were mediated through phosphorylation of Src and EGFR via the formation of Src/EGFR complex which was attenuated by PP1 and AG1478. Moreover, BK-induced p42/p44 MAPK and Akt activation was mediated through EGFR transactivation, which was diminished by the inhibitors of MMP-2/9 and heparin-binding EGF-like factor (HB-EGF). Finally, increased nuclear translocation of Akt and p42/p44 MAPK turns on early gene expression leading to cell proliferation. These results suggest that BK-induced cell proliferation is mediated through c-Src-dependent transactivation of EGFR via MMP2/9-dependent pro-HB-EGF shedding linking to activation of Akt and p42/p44 MAPK in corneal fibroblasts. Copyright © 2011 Wiley Periodicals, Inc.

  6. Human thymic epithelial cells express functional HLA-DP molecules

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

    T lymphocytes, we examined whether human thymic epithelial cells (TEC) expressed HLA-DP molecules. We present evidence that TEC obtained from short time culture express low but significant levels of HLA-DP molecules. The expression of HLA-DP molecules was comparable to or higher than the expression...... of HLA-DP allospecific primed lymphocyte typing (PLT) CD4 T cell lines. IFN-gamma treatment strongly upregulated the HLA-DP allospecific PLT responses whereas other PLT responses remained largely unchanged. In conclusion, these data indicate that human thymus epithelial cells express significant levels...

  7. Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells.

    Science.gov (United States)

    Bosshardt, D D; Nanci, A

    1998-02-01

    After crown formation, the enamel organ reorganizes into Hertwig's epithelial root sheath (HERS). Although it is generally accepted that HERS plays an inductive role during root formation, it also has been suggested that it may contribute enamel-related proteins to cementum matrix. By analogy to the enamel-free area (EFA) in rat molars, in which epithelial cells express not only enamel proteins but also "typical" mesenchymal matrix constituents, it has been proposed that HERS cells may also have the potential to produce cementum proteins. To test this hypothesis, we examined the nature of the first matrix layer deposited along the cervical portion of root dentin and the characteristics of the associated cells. Rat molars were processed for postembedding colloidal gold immunolabeling with antibodies to amelogenin (AMEL), ameloblastin (AMBN), bone sialoprotein (BSP), and osteopontin (OPN). To minimize the possibility of false-negative results, several antibodies to AMEL were used. The labelings were compared with those obtained at the EFA. Initial cementum matrix was consistently observed at a time when epithelial cells from HERS covered most of the forming root surface. Cells with mesenchymal characteristics were rarely seen in proximity to the matrix. Both the EFA matrix and initial cementum exhibited collagen fibrils and were intensely immunoreactive for BSP and OPN. AMEL and AMBN were immunodetected at the EFA but not over the initial cementum proper. These two proteins were, however, present at the cervical-most portion of the root where enamel matrix extends for a short distance between dentin and cementum. These data suggest that epithelial cells along the root surface are likely responsible for the deposition of the initial cementum matrix and therefore, like the cells at the EFA, may be capable of producing mesenchymal proteins.

  8. Photo-cross-linking of amniotic membranes for limbal epithelial cell cultivation

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Jui-Yang, E-mail: jylai@mail.cgu.edu.tw

    2014-12-01

    In the present study, we developed photo-cross-linked amniotic membrane (AM) as a limbal stem cell niche. After ultraviolet (UV) irradiation for varying time periods, the biological tissues were studied by determinations of cross-linking structure, degradability, and nutrient permeation ability. Our results showed that the number of cross-links per unit mass of AM significantly increased with increasing illumination time from 5 to 50 min. However, the cross-link formation was inhibited by longer irradiation time (i.e., 150 min), probably due to the scission of tissue collagen chains through irradiation. The biological stability and matrix permeability of photo-cross-linked AM materials strongly depended on their cross-linking densities affected by the UV irradiation. In vitro biocompatibility studies including cell viability and pro-inflammatory gene expression analyses demonstrated that, irrespective of the irradiation time employed, the physically cross-linked biological tissues exhibited negligible cytotoxicity and similar interleukin-6 (IL-6) mRNA levels. The data clearly indicate that these AM matrices do not cause potential harm to the corneal epithelial cells. After the growth of limbal epithelial cells (LECs) on AM substrates, Western blot analyses were conducted to examine the expression of ABCG2. It was found that the ability of UV-irradiated AM to maintain the undifferentiated precursor cell phenotype was significantly enhanced with increasing extent of photo-cross-linking. In summary, the UV irradiation time may have a profound influence on the fabrication of photo-cross-linked AM matrices for LEC cultivation. - Highlights: • We report the development of photo-cross-linked AM as a limbal stem cell niche. • Cross-linked structure of tissue materials was controlled by UV irradiation time. • Biostability and matrix permeability of AM depended on cross-linking density. • All the studied photo-cross-linked AM showed good in vitro biocompatibility.

  9. 应用羊膜上皮干细胞微环境培养人角膜内皮细胞的研究%Microenvironment of amniotic epithelium cells enhances the proliferation of human corneal endothelial cells

    Institute of Scientific and Technical Information of China (English)

    王忠浩; 陈玮; 宋莉; 沙翔垠; 梁轩伟

    2013-01-01

    Objective To establish an effective method to enhance the proliferation of human corneal endothelial cells (HCECs). Methods The culture conditions of HCEC were optimized by utilizing the totipotent characteristics of human amniotic membrane epithelial stem cells (HAEC) to establish the optimal culture microenvironment of HAEC to promote the proliferation of HCEC. The morphology of HCEC was observed by using phase-contrast microscope and transmission electron microscope. MTT assay and Giemsa staining were performed to detect the proliferation of HCEC. The rate of apoptotic cells was investigated by using Hoechst33342 staining assay. Results Compared to the corneal endothelial cells medium (CEM), the microenvironment containing 20% HAEC-conditioned medium and HAEC-HCEC co-culture microenvironment could promote the proliferation of HCEC and could reduce the apoptosis of HCEC. The cells in HAEC-HCEC microenvironment group could be passaged 4 times without lossing their polygonal appearance. Conclusion The HAEC microenvironment could effectively enhance the proliferation of HCEC, maintain the morphology of HCEC, and inhibit the process of apoptosis of HCEC.%目的:建立一种利用羊膜上皮干细胞(human amniotic membrane epithelial cell,HAEC)微环境培养人角膜内皮细胞(human corneal endothelial cells,HCEC)的方法.方法:制备羊膜上皮干细胞微环境培养HCEC,并探讨诱导HCEC增殖的最佳培养微环境,倒置相差显微镜和透射电镜观察培养过程中细胞的形态学变化,MTT和Giemsa染色观察细胞增殖情况,Hoechst33342检测凋亡细胞比例.结果:在HCEC基本培养液(corneal endothelial cell medium,CEM)的基础上添加20% HAEC上清、HAEC-HCEC的微环境可促进HCEC的增殖,减少凋亡,细胞传代能力显著增强,HAEC-HCEC组传至4代仍保持多角形的内皮细胞形态.结论:羊膜上皮干细胞微环境培养可有效提高HCEE的增殖能力,更好地维持HCEC的形态,并能抑制其凋亡进程.

  10. Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells

    Science.gov (United States)

    Chen, Qike K.; Lee, KangAe; Radisky, Derek C.; Nelson, Celeste M.

    2013-01-01

    Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland. PMID:23660532

  11. Effect of Helicobacter pylori on gastric epithelial cells

    Science.gov (United States)

    Alzahrani, Shatha; Lina, Taslima T; Gonzalez, Jazmin; Pinchuk, Irina V; Beswick, Ellen J; Reyes, Victor E

    2014-01-01

    The gastrointestinal epithelium has cells with features that make them a powerful line of defense in innate mucosal immunity. Features that allow gastrointestinal epithelial cells to contribute in innate defense include cell barrier integrity, cell turnover, autophagy, and innate immune responses. Helicobacter pylori (H. pylori) is a spiral shape gram negative bacterium that selectively colonizes the gastric epithelium of more than half of the world’s population. The infection invariably becomes persistent due to highly specialized mechanisms that facilitate H. pylori’s avoidance of this initial line of host defense as well as adaptive immune mechanisms. The host response is thus unsuccessful in clearing the infection and as a result becomes established as a persistent infection promoting chronic inflammation. In some individuals the associated inflammation contributes to ulcerogenesis or neoplasia. H. pylori has an array of different strategies to interact intimately with epithelial cells and manipulate their cellular processes and functions. Among the multiple aspects that H. pylori affects in gastric epithelial cells are their distribution of epithelial junctions, DNA damage, apoptosis, proliferation, stimulation of cytokine production, and cell transformation. Some of these processes are initiated as a result of the activation of signaling mechanisms activated on binding of H. pylori to cell surface receptors or via soluble virulence factors that gain access to the epithelium. The multiple responses by the epithelium to the infection contribute to pathogenesis associated with H. pylori. PMID:25278677

  12. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    Science.gov (United States)

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  13. Correlation between Corneal Endothelial Cell Loss and Location of Phacoemulsification Incision

    Directory of Open Access Journals (Sweden)

    Hamid Gharaee

    2011-01-01

    Full Text Available Purpose: To assess the relationship between corneal endothelial cell loss after phacoemulsification and the location of the clear corneal incision. Methods: A total of 92 patients (92 eyes with senile cataracts who met the study criteria were included in this cross sectional study and underwent phacoemulsification. The incision site was determined based on the steep corneal meridian according to preoperative keratometry. Endothelial cell density was measured using specular microscopy in the center and 3 mm from the center of the cornea in the meridian of the incisions (temporal, superior, and superotemporal. Phacoemulsification was performed by a single surgeon using the phaco chop technique through a 3.2 mm clear cornea incision. Endothelial cell loss (ECL was evaluated 1 week, and 1 and 3 months postoperatively. Results: At all time points during follow-up, ECL was comparable among the 3 incision sites, both in the central cornea and in the meridian of the incision (P > 0.05 for all comparisons. However, 3 months postoperatively, mean central ECL with superior incisions and mean sectoral ECL with temporal incisions were slightly higher. Superotemporal incisions entailed slightly less ECL than the other 2 groups. Overall, one month after surgery, mean central ECL was 10.8% and mean ECL in the sector of the incisions was 14.0%. Axial length and effective phaco time (EFT were independent predictors of postoperative central ECL (P values 0.005 and < 0.0001, respectively. Conclusion: A superotemporal phacoemulsification incision may entail less ECL as compared to other incisions (although not significantly different. The amount of central ECL may be less marked in patients with longer axial lengths and with procedures utilizing less EFT.

  14. Aloe vera: an in vitro study of effects on corneal wound closure and collagenase activity.

    Science.gov (United States)

    Curto, Elizabeth M; Labelle, Amber; Chandler, Heather L

    2014-11-01

    To evaluate the in vitro effects of an aloe vera solution on (i) the viability and wound healing response of corneal cells and (ii) the ability to alter collagenase and gelatinase activities. Primary cultures of corneal epithelial cells and fibroblasts were prepared from grossly normal enucleated canine globes and treated with an aloe solution (doses ranging from 0.0-2 mg/mL). Cellular viability was evaluated using a colorimetric assay. A corneal wound healing model was used to quantify cellular ingrowth across a defect made on the confluent surface. Anticollagenase and antigelatinase activities were evaluated by incubating a bacterial collagenase/gelatinase with aloe solution (doses ranging from 0.0-500 μg/mL) and comparing outcome measures to a general metalloproteinase inhibitor, 1, 10-phenanthroline, and canine serum (doses ranging from 0.0-100%). None of the concentrations of aloe solution tested significantly affected the viability of corneal epithelial cells or fibroblasts. Concentrations ≤175 μg/mL slightly accelerated corneal epithelial cell wound closure; this change was not significant. Concentrations ≥175 μg/mL significantly (P ≤ 0.001) slowed the rate of corneal fibroblast wound closure, while aloe concentrations Aloe solution did not alter the ability for collagenase to degrade gelatin or collagen Type I but increased the ability for collagenase to degrade Type IV collagen. Although additional experiments are required, lower concentrations of aloe solution may be beneficial in healing of superficial corneal wounds to help decrease fibrosis and speed epithelialization. An increase in collagenase activity with aloe vera warrants further testing before considering in vivo studies. © 2014 American College of Veterinary Ophthalmologists.

  15. Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

    Science.gov (United States)

    Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

    2015-12-01

    The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  16. Superficial keratectomy and topical mitomycin C as therapy for a corneal squamous cell carcinoma in a dog.

    Science.gov (United States)

    Karasawa, K; Matsuda, H; Tanaka, A

    2008-04-01

    A 10-year-old female West Highland white terrier was presented with refractory hyperplastic keratitis of the left cornea of one month's duration. At this time, a vascularised and rough lesion 5 mm in diameter was observed on the left cornea. No other abnormality was recognised on the affected eye. The corneal neoplasm was surgically removed and histologically diagnosed as a squamous cell carcinoma. For two months after the surgery, 0.04 percent mitomycin C (MMC) eye drops were applied as adjuvant chemotherapy. Primary corneal squamous cell carcinoma with no history of keratoconjunctivitis sicca is rare in dogs. In the present report, surgical removal of the neoplasm was combined with the topical administration of the anticancer drug mitomycin C and a good prognosis was obtained. The result indicates that the combination treatment used in this case may be an appropriate therapeutic choice for corneal squamous cell carcinoma in dogs.

  17. Collective Movement of Epithelial Cells on a Collagen Gel Substrate

    OpenAIRE

    Haga, Hisashi; Irahara, Chikako; KOBAYASHI, Ryo; Nakagaki, Toshiyuki; Kawabata, Kazushige

    2004-01-01

    Collective cell movement acts as an efficient strategy in many physiological events, including wound healing, embryonic development, and morphogenesis. We found that epithelial cells (Madin-Darby canine kidney cell) migrated collectively along one direction on a collagen gel substrate. Time-lapse images of Madin-Darby canine kidney cells cultured on type-I collagen gels and glass substrates were captured by phase contrast microscopy equipped with an incubation system. On the gel substrate, th...

  18. Role of p53 Mammary Epithelial Cell Senescence

    Science.gov (United States)

    2005-05-01

    AD Award Number: DAMD17-02-1-0509 TITLE: Role of p53 Mammary Epithelial Cell Senescence PRINCIPAL INVESTIGATOR: Goberdhan P. Dimri, Ph.D. CONTRACTING ...type and However, Mucl , K-18, and ASMA were not expressed in luminal cell type groups [12,68]. Interestingly, a significant cells present in...13,17,27], the has also attracted a great interest in the field of breast cancer candidate mammary stem cells appear to be ESA+, Mucl -, research, and

  19. Coculture of dorsal root ganglion neurons and differentiated human corneal stromal stem cells on silk-based scaffolds.

    Science.gov (United States)

    Wang, Siran; Ghezzi, Chiara E; White, James D; Kaplan, David L

    2015-10-01

    Corneal tissue displays the highest peripheral nerve density in the human body. Engineering of biomaterials to promote interactions between neurons and corneal tissue could provide tissue models for nerve/cornea development, platforms for drug screening, as well as innovative opportunities to regenerate cornea tissue. The focus of this study was to develop a coculture system for differentiated human corneal stromal stem cells (dhCSSCs) and dorsal root ganglion neurons (DRG) to mimic the human cornea tissue interactions. Axon extension, connectivity, and neuron cell viability were studied. DRG neurons developed longer axons when cocultured with dhCSSCs in comparison to neuron cultures alone. To assess the mechanism involved in the coculture response, nerve growth factors (NGF) secreted by dhCSSCs including NGF, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and neurotrophin-3 were characterized with greater focus on BDNF secretion. DhCSSCs also secreted collagen type I, an extracellular matrix molecule favorable for neuronal outgrowth. This coculture system provides a slowly degrading silk matrix to study neuronal responses in concert with hCSSCs related to innervation of corneal tissue with utility toward human corneal nerve regeneration and associated diseases. © 2015 Wiley Periodicals, Inc.

  20. Mitochondrial oxygen metabolism in primary human lens epithelial cells: Association with age, diabetes and glaucoma.

    Science.gov (United States)

    Kubota, M; Shui, Y B; Liu, M; Bai, F; Huang, A J; Ma, N; Beebe, D C; Siegfried, C J

    2016-08-01

    The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells. Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U test and multivariate regression analysis. Primary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21±1.037 vs. 2.86±1.383 fmol/min/cell; pOCR than non-diabetic patients (2.02±0.911 vs. 2.79±1.332fmol/min/cell; pOCR than non-glaucoma patients (2.27±1.19 vs. 2.83±1.286 fmol/min/cell; pOCR, independent of other variables. Gender and race had no significant effect on OCR. The lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma could contribute to cataract development. Diabetes and glaucoma are particularly important factors associated with decreased OCR, independent of age. Ongoing studies are examining pO2 at the anterior surface of the lens in vivo and oxygen consumption in the patient's LECs. Copyright © 2016 Elsevier

  1. Efeito do mel e do soro autólogo na cicatrização do epitélio corneano em coelhos Effect of honey and autologous serum on corneal epithelial healing in rabbits

    Directory of Open Access Journals (Sweden)

    Gustavo Ricci Malavazzi

    2005-06-01

    Full Text Available OBJETIVO: Avaliar a eficácia de substâncias consideradas estimulantes da cicatrização, como o mel puro e o soro autólogo a 20% na cicatrização do epitélio corneal de coelhos. MÉTODOS: Foi realizada a remoção do epitélio corneal de dois grupos de coelhos que receberam a instilação de solução de mel puro (G1 ou soro autólogo (G2 a cada 4 horas. O olho contralateral foi usado como controle e submetido ao mesmo procedimento de remoção do epitélio, recebendo a instilação de BSS®. A área de desepitelização corneal foi avaliada 12, 24 e 48 horas após a indução do defeito epitelial. RESULTADOS: Os grupos estudados foram estatisticamente semelhantes: mel (48 horas e controle (48 horas pPURPOSE: To evaluate the efficacy of pure honey and 20% autologous serum and BSS® in corneal epithelial healing in rabbits after 48 hours. METHODS: All solutions were applied after an epithelial removal of 13-millimeters diameter area. Areas of epithelial healing were studied at 12, 24 and 48 hours. The eyes were treated every four hours during 2 days. All treated eyes were assigned to a control group (contralateral eye treated with a balanced saline solution. RESULTS: All studied groups were not significantly differents. In group one, the eyes treated with honey and the control were similar (p<0.87. In the second group the eyes treated with autologos serum and the control presented no difference in the mean score (p<0.072. CONCLUSION: Corneal epithelial healing in rabbits did not show improvement after application of either honey or autologous serum. It was possible to stabilish that the autologous serum treated eyes were clinicaly better than the control group but without statistical significance.

  2. Corneal endothelial cell loss during phacoemulsification: bevel-up versus bevel-down phaco tip.

    Science.gov (United States)

    Faramarzi, Amir; Javadi, Mohammad Ali; Karimian, Farid; Jafarinasab, Mohammad Reza; Baradaran-Rafii, Alireza; Jafari, Fariba; Yaseri, Mehdi

    2011-11-01

    To compare corneal endothelial cell loss during cataract extraction by phacoemulsification with 2 different phaco-tip positions. Ophthalmic Research Center and Department of Ophthalmology, Labbafinejad Medical Center, Shahid Beheshti Medical University, Tehran, Iran. Randomized clinical trial. Eyes scheduled for cataract extraction were randomly assigned stop-and-chop phacoemulsification with the phaco tip in the conventional bevel-up position or with the phaco tip in the bevel-down position. During surgery, the effective phacoemulsification time (EPT) was recorded. Preoperative endothelial cell parameters were compared with measurements taken 3 months postoperatively. Each group comprised 30 eyes (30 patients). There were no statistically significant differences in age, sex, anterior chamber depth, axial length, or EPT between the 2 groups. The mean preoperative endothelial cell density (ECD) was 2544 cells/mm(2) ± 64 (SD) in the bevel-up group and 2471 ± 59 cells/mm(2) in the bevel-down group (P=.610). Postoperatively, both groups had a significant decrease in ECD. The mean endothelial cell loss was 5.9% in the bevel-up group and 13.6% in the bevel-down group (P=.012). The percentage of hexagonal cells and coefficient of variation in cell size were not different between the 2 groups preoperatively or postoperatively; however, after surgery, there was a significant decrease in the percentage of hexagonal cells in both groups. Corneal endothelial cell loss during phacoemulsification was significantly higher when the phaco tip was in the bevel-down position than in the conventional bevel-up position. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2011 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  3. Limbal Stem Cell Transplantation for Gelatinous Drop-like Corneal Dystrophy

    Directory of Open Access Journals (Sweden)

    Hossein Movahedan

    2013-01-01

    Full Text Available Purpose: To report the outcomes of allograft limbal stem cell transplantation for recurrent gelatinous drop-like corneal dystrophy (GDLD. Methods: In this non-comparative interventional case series, 4 eyes of 3 consecutive patients with recurrent GDLD underwent allograft limbal stem cell transplantation. Two eyes underwent concomitant penetrating keratoplasty while the other two underwent simultaneous superficial keratectomy. Main outcome measures were best spectacle corrected visual acuity, intraocular pressure and corneal clarity. Results: Patient age ranged from 28 to 63 years. Mean follow-up after surgery was 23±10 (median, 22; range, 12-36 months. Mean visual acuity improved from 2.70±0.61 logMAR preoperatively to 1.05±0.06 logMAR at final postoperative visit (P=0.066. Intraocular pressure was normal in all eyes at baseline and remained within normal limits at all postoperative visits. All corneas remained smooth and clear during the follow-up period with no episode of rejection or recurrence. All patients maintained ambulatory vision until final follow-up. Conclusion: The present study provides further evidence that limbal stem cell transplantation may be an effective therapeutic alternative in patients with GDLD.

  4. The Effect of Fibronectin on Re-epithelialization of Rabbits Cornea after Alkali Burn

    Institute of Scientific and Technical Information of China (English)

    1994-01-01

    The authors found experimentally that (1) fibronectin enhanced the healing of rabbit corneal epithelium after alkali burn and prevented the secondary breakdown; (2) it rapidly deposited on the denuded basement membrane to disappear as epithelial cells slided over, and (3) ultrastructurally, the neighbouring epithelial cells became flattened, with filopodia at the advancing edge, and extended to the wounded areas at 24 hours after the burn. However, the epithelial defects recurred 72 hours after the burn...

  5. Tiotropium attenuates IL-13-induced goblet cell metaplasia of human airway epithelial cells

    NARCIS (Netherlands)

    Kistemaker, Loes E. M.; Hiemstra, Pieter S.; Bos, I. Sophie T.; Bouwman, Susanne; van den Berge, Maarten; Hylkema, Machteld N.; Meurs, Herman; Kerstjens, Huib A. M.; Gosens, Reinoud

    2015-01-01

    BACKGROUND: It has been shown that acetylcholine is both a neurotransmitter and acts as a local mediator, produced by airway cells including epithelial cells. In vivo studies have demonstrated an indirect role for acetylcholine in epithelial cell differentiation. Here, we aimed to investigate direct

  6. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    Science.gov (United States)

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

  7. Microarray analysis of human epithelial cell responses to bacterial interaction.

    Science.gov (United States)

    Mans, Jeffrey J; Lamont, Richard J; Handfield, Martin

    2006-09-01

    Host-pathogen interactions are inherently complex and dynamic. The recent use of human microarrays has been invaluable to monitor the effects of various bacterial and viral pathogens upon host cell gene expression programs. This methodology has allowed the host response transcriptome of several cell lines to be studied on a global scale. To this point, the great majority of reports have focused on the response of immune cells, including macrophages and dendritic cells. These studies revealed that the immune response to microbial pathogens is tailored to different microbial challenges. Conversely, the paradigm for epithelial cells has--until recently--held that the epithelium mostly served as a relatively passive physical barrier to infection. It is now generally accepted that the epithelial barrier contributes more actively to signaling events in the immune response. In light of this shift, this review will compare transcriptional profiling data from studies that involved host-pathogen interactions occurring with epithelial cells. Experiments that defined both a common core response, as well as pathogen-specific host responses will be discussed. This review will also summarize the contributions that transcriptional profiling analysis has made to our understanding of bacterial physio-pathogensis of infection. This will include a discussion of how host transcriptional responses can be used to infer the function of virulence determinants from bacterial pathogens interacting with epithelial mucosa. In particular, we will expand upon the lessons that have been learned from gastro-intestinal and oral pathogens, as well as from members of the commensal flora.

  8. Growth and differentiation of human lens epithelial cells in vitro on matrix

    Science.gov (United States)

    Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.; Aragon, G.; Lin, S. P.; Lui, G.; Polansky, J. R.

    2000-01-01

    PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

  9. Keratocyte loss in corneal infection through apoptosis: a histologic study of 59 cases

    Science.gov (United States)

    Vemuganti, Geeta K; Reddy, Kishore; Iftekhar, Ghazala; Garg, Prashant; Sharma, Savitri

    2004-01-01

    Background Keratocyte loss by apoptosis following epithelial debridement is a well-recognized entity. In a study of corneal buttons obtained from patients of corneal ulcer undergoing therapeutic keratoplasty, we observed loss of keratocytes in the normal appearing corneal stroma, surrounding the zone of inflammation. Based on these observations, we hypothesized that the cell loss in the inflammatory free zone of corneal stroma is by apoptosis that could possibly be a non-specific host response, independent of the nature of infectious agent. Methods To test our hypothesis, in this study, we performed Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Label