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Sample records for epithelial corneal cells

  1. Ciliary neurotrophic factor promotes the activation of corneal epithelial stem/progenitor cells and accelerates corneal epithelial wound healing.

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    Zhou, Qingjun; Chen, Peng; Di, Guohu; Zhang, Yangyang; Wang, Yao; Qi, Xia; Duan, Haoyun; Xie, Lixin

    2015-05-01

    Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. © 2014 AlphaMed Press.

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

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    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.

  3. Protective Effects of Trehalose on the Corneal Epithelial Cells

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    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.

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

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    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. Copyright © 2012 John Wiley & Sons, Ltd.

  5. Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

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    Bhavani S. Kowtharapu

    2018-02-01

    Full Text Available Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM on corneal epithelial cell function along with substance P (SP. Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK, paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained

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

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    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.

  7. Cellular toxicity of calf blood extract on human corneal epithelial cells in vitro.

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    Park, Young Min; Kim, Su Jin; Han, Young Sang; Lee, Jong Soo

    2015-01-01

    To investigate the biologic effects of the calf blood extract on corneal epithelial cells in vitro. The effects on corneal epithelial cells were evaluated after 1, 4, 12, and 24 h of exposure to various concentrations of calf blood extract (3, 5, 8 and 16%). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was performed to measure levels of cellular metabolic activity. The lactate dehydrogenase (LDH) assay was performed to determine the extent of cellular damage. Cellular morphology was examined using phase-contrast microscopy. The scratch wound assay was performed to quantify the migration of corneal epithelial cells. At the 3 and 5% concentrations of calf blood extract, MTT values were similar to those observed in the control group. However, at a concentration of 8 and 16%, cellular metabolic activity was significantly decreased after 4 h of exposure to calf blood extract. After 12 h of exposure to 8 and 16% concentrations of calf blood extract, LDH activity and cellular morphological damage to the corneal epithelial cells were significantly increased. There was no evidence of cellular migration after 12 h exposure to 5% or higher concentration of calf blood extract because of cellular toxicity. Compared with normal corneal epithelial cells, the cellular activity was decreased, and toxicity was increased after over 12 h of exposure to more than 5% concentration of calf blood extract. Further clinical studies will be necessary to determine the optimal concentration and exposure time for the topical application of eye drops containing calf blood extract.

  8. Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits

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    Kim, Tae-Hyun; Park, Young-Woo; Ahn, Jae-Sang; Ahn, Jeong-Taek; Kim, Se-Eun; Jeong, Man-Bok; Seo, Min-Su; Kang, Kyung-Sun

    2013-01-01

    This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits. PMID:23388445

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

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    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.

  10. Human corneal epithelial subpopulations

    DEFF Research Database (Denmark)

    Søndergaard, Chris Bath

    2013-01-01

    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...... 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 differentiation pathway in human corneal epithelium according to an optimized protocol for maintenance of expression profiles...

  11. Glucocorticoid action in human corneal epithelial cells establishes roles for corticosteroids in wound healing and barrier function of the eye.

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    Kadmiel, Mahita; Janoshazi, Agnes; Xu, Xiaojiang; Cidlowski, John A

    2016-11-01

    Glucocorticoids play diverse roles in almost all physiological systems of the body, including both anti-inflammatory and immunosuppressive roles. Synthetic glucocorticoids are one of the most widely prescribed drugs and are used in the treatment of conditions such as autoimmune diseases, allergies, ocular disorders and certain types of cancers. In the interest of investigating glucocorticoid actions in the cornea of the eye, we established that multiple cell types in mouse corneas express functional glucocorticoid receptor (GR) with corneal epithelial cells having robust expression. To define glucocorticoid actions in a cell type-specific manner, we employed immortalized human corneal epithelial (HCE) cell line to define the glucocorticoid transcriptome and elucidated its functions in corneal epithelial cells. Over 4000 genes were significantly regulated within 6 h of dexamethasone treatment, and genes associated with cell movement, cytoskeletal remodeling and permeability were highly regulated. Real-time in vitro wound healing assays revealed that glucocorticoids delay wound healing by attenuating cell migration. These functional alterations were associated with cytoskeletal remodeling at the wounded edge of a scratch-wounded monolayer. However, glucocorticoid treatment improved the organization of tight-junction proteins and enhanced the epithelial barrier function. Our results demonstrate that glucocorticoids profoundly alter corneal epithelial gene expression and many of these changes likely impact both wound healing and epithelial cell barrier function. Published by Elsevier Ltd.

  12. Inhibition of UV-B induced apoptosis in corneal epithelial cells by potassium channel modulators.

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    Ubels, John L; Schotanus, Mark P; Bardolph, Susan L; Haarsma, Loren D; Koetje, Leah R; Louters, Julienne R

    2010-02-01

    The goal of this study was to determine whether prevention of K(+) loss can protect human corneal-limbal epithelial (HCLE) cells from UV-B induced apoptosis. Immunostaining for activated caspase-3 of HCLE cells exposed to 150-200 mJ/cm(2) UV-B demonstrated induction of apoptosis 6 h after exposure. The number of apoptotic cells was decreased by incubation in medium with 25 or 100 mM K(+). If this protection is due to a reduction of UV-induced K(+) loss then K(+) channel blockers should also protect HCLE cells from UV-B. Caspase-8 activity induced by exposure to UV-B at 150 mJ/cm(2) was significantly reduced when the cells were incubated in 0.3 microM BDS-I or 0.05-1 mM quinidine. Caspase-3 was also activated by UV-B and a reduction in activity was observed after incubation in 0.1-0.3 microM BDS-I and 0.1-1 mM quinidine. Induction of DNA fragmentation, as measured by the TUNEL assay, was decreased by treatment with 0.3 microM BDS-I and 0.01-0.05 mM quinidine. Patch-clamp recording showed activation of K(+) channels after exposure to UV-B and a decrease in outward K(+) current was observed following application of BDS-I. Quinidine did not block K(+) currents in HCLE cells, suggesting that the protective effect of quinidine occurs by a mechanism other than via K(+) channels. The effect of the K(+) channel blocker BDS-1 on HCLE cells exposed to UV-B confirms that preventing K(+) efflux protects corneal epithelial cells from apoptosis. This suggests the elevated [K(+)] in tears may protect the corneal epithelium from effects of ambient UV-B. Copyright 2009 Elsevier Ltd. All rights reserved.

  13. Inhibition of UV-B Induced Apoptosis in Corneal Epithelial Cells by Potassium Channel Modulators

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    Ubels, John L.; Schotanus, Mark P.; Bardolph, Susan L.; Haarsma, Loren D.; Koetje, Leah R.; Louters, Julienne R.

    2009-01-01

    The goal of this study was to determine whether prevention of K+ loss can protect human corneal-limbal epithelial (HCLE) cells from UV-B induced apoptosis. Immunostaining for activated caspase-3 of HCLE cells exposed to 150 – 200 mJ/cm2 UV-B demonstrated induction of apoptosis 6 hrs after exposure. The number of apoptotic cells was decreased by incubation in medium with 25 or 100 mM K+. If this protection is due to a reduction of UV induced K+ loss then K+ channel blockers should also protect HCLE cells from UV-B. Caspase-8 activity induced by exposure to UV-B at 150 mJ/cm2 was significantly reduced when the cells were incubated in 0.3 µM BDS-I or 0.05–1 mM quinidine. Caspase-3 was also activated by UV-B and a reduction in activity was observed after incubation in 0.1–0.3 µM BDS-I and 0.1–1mM quinidine. Induction of DNA fragmentation, as measured by the TUNEL assay, was decreased by treatment with 0.3 µM BDS-I and 0.01–0.05 mM quinidine. Patch-clamp recording showed activation of K+ channels after exposure to UV-B and a decrease in outward K+ current was observed following application of BDS-I. Quinidine did not block K+ currents in HCLE cells, suggesting that the protective effect of quinidine occurs by a mechanism other than via K+ channels. The effect of the K+ channel blocker BDS-1 on HCLE cells exposed to UV-B confirms that preventing K+ efflux protects corneal epithelial cells from apoptosis. This suggests the elevated [K+] in tears may protect the corneal epithelium from effects of ambient UV-B. PMID:19874821

  14. Nuclear ferritin in corneal epithelial cells: tissue-specific nuclear transport and protection from UV-damage.

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    Linsenmayer, Thomas F; Cai, Cindy X; Millholland, John M; Beazley, Kelly E; Fitch, John M

    2005-03-01

    We have identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form that is indistinguishable from the cytoplasmic form of ferritin found in other cell types. Thus it most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and other tissues were UV irradiated, and damage to DNA was detected by an in situ 3'-end labeling assay. Consistent with the hypothesis, corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than the other cells types examined. However, when the expression of nuclear ferritin was inhibited the cells now became much more susceptible to UV-induced DNA damage. Since ferritin is normally cytoplasmic, corneal epithelial cells must have a mechanism that effects its nuclear localization. We have determined that this involves a nuclear transport molecule which binds to ferritin and carries it into the nucleus. This transporter, which we have termed ferritoid for its similarity to ferritin, has at least two domains. One domain is ferritin-like and is responsible for binding the ferritin; the other domain contains a nuclear localization signal that is responsible for effecting the nuclear transport. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage. In addition, since ferritoid is structurally similar to ferritin, it may represent an example of a nuclear transporter that evolved from the molecule it transports (i.e., ferritin).

  15. Establishment of an untransfected human corneal epithelial cell line and its biocompatibility with denuded amniotic membrane.

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    Fan, Ting-Jun; Xu, Bin; Zhao, Jun; Yang, Hong-Shou; Wang, Rui-Xin; Hu, Xiu-Zhong

    2011-01-01

    To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM). The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors, yielding an HCEP cell line which was its growth performance, chromosome morphology, tumorigenicity and expression of marker proteins analyzed. In addition, the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light, electron and slit-lamp microscopies. HCEP cells proliferated to confluence in 3 weeks, which have been subcultured to passage 160. A continuous untransfected HCEP cell line, designated as utHCEPC01, was established with a population doubling time of 45.42 hours as was determined at passage 100. The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3. They, with no tumorigenicity, formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture, maintained expression of marker proteins including keratin 3 and integrin β1 and attached tightly to dAMs. The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original. An untransfected and non-tumorigenic HCEP cell line was established in this study. The cells maintained expression of marker proteins. The cell line was biocompatible with dAM. It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP, promising for the treatment of diseases caused by corneal epithelial disorders.

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

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    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.

  17. Elevated extracellular K+ inhibits apoptosis of corneal epithelial cells exposed to UV-B radiation.

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    Singleton, Katherine R; Will, David S; Schotanus, Mark P; Haarsma, Loren D; Koetje, Leah R; Bardolph, Susan L; Ubels, John L

    2009-08-01

    The goal of this study was to determine if the high [K(+)] in tears, 20-25 mM, serves to protect corneal epithelial cells from going into apoptosis after exposure to ambient UV-B radiation. Human corneal-limbal epithelial (HCLE) cells in culture were exposed to UV-B at doses of 50-200 mJ/cm(2) followed by measurement of K(+) channel activation and activity of apoptotic pathways. Patch-clamp recording showed activation of K(+) channels after UV-B exposure at 80 mJ/cm(2) or 150 mJ/cm(2) and a decrease in UV-induced K(+) efflux with increasing [K(+)](o). The UV-activated current was partially blocked by the specific K(+) channel blocker, BDS-1. DNA fragmentation, as measured by the TUNEL assay, was induced after exposure to UV-B at 100-200 mJ/cm(2). DNA fragmentation was significantly decreased when cells were incubated in 25, 50 or 100mM K(o)(+) after exposure to UV-B. The effector caspase, caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm(2), but there was a significant decrease in activation when the cells were incubated in 25, 50 or 100mM K(o)(+) following exposure to UV-B. A decrease in mitochondrial potential, a possible activator of caspase-3, occurred after exposure to UV-B at 100-200 mJ/cm(2). This decrease in mitochondrial potential was prevented by 100mM K(o)(+); however, 25 or 50mM K(o)(+) provided minimal protection. Caspase-9, which is in the pathway from mitochondrial potential change to caspase-3 activation, showed little activation by UV-B radiation. Caspase-8, an initiator caspase that activates caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm(2), and this UV-activation was significantly reduced by 25-100mM K(o)(+). The data show that the physiologically relevant [K(+)](o) of 25 mM can inhibit UV-B induced activation of apoptotic pathways. This suggests that the relatively high [K(+)] in tears reduces loss of K(+) from corneal epithelial cells in response to UV exposure, thereby contributing to the protection of the ocular

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

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    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.

  19. Effects of organophosphorus flame retardant TDCPP on normal human corneal epithelial cells: Implications for human health.

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    Xiang, Ping; Liu, Rong-Yan; Li, Chao; Gao, Peng; Cui, Xin-Yi; Ma, Lena Q

    2017-11-01

    Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is one of the most detected organophosphorus flame retardants (OPFRs) in the environment, especially in indoor dust. Continuous daily exposure to TDCPP-containing dust may adversely impact human cornea. However, its detrimental effects on human corneal epithelium are largely unknown. In this study, we investigated the cell apoptosis in normal human corneal epithelial cells (HCECs) after TDCPP exposure and elucidated the underlying molecular mechanisms. Our data indicated a dose-dependent decrease of cell viability after TDCPP exposure with LC 50 at 202 μg/mL. A concentration-dependent apoptotic sign was observed in HCECs after exposing to ≥2 μg/mL TDCPP. Endoplasmic reticulum stress induction was evidenced by up-regulation of its biomarker genes (ATF-4, CHOP, BiP, and XBP1). Furthermore, alternation of Bcl-2/Bax expression, mitochondrial membrane potential loss, cellular ATP content decrease, and caspase-3 and -9 activity increase were observed after exposing to 2 or 20 μg/mL TDCPP. Taken together, the data implicated the involvement of endoplasmic reticulum stress in TDCPP-induced HCEC apoptosis, probably mediated by mitochondrial apoptotic pathway. Our findings showed TDCPP exposure induced toxicity to human cornea. Due to TDCPP's presence at high levels in indoor dust, further study is warranted to evaluate its health risk on human corneas. Published by Elsevier Ltd.

  20. 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

  1. The impact of type 1 diabetes mellitus on corneal epithelial nerve morphology and the corneal epithelium.

    Science.gov (United States)

    Cai, Daniel; Zhu, Meifang; Petroll, W Matthew; Koppaka, Vindhya; Robertson, Danielle M

    2014-10-01

    Diabetic corneal neuropathy can result in chronic, sight-threatening corneal pathology. Although the exact etiology is unknown, it is believed that a reduction in corneal sensitivity and loss of neurotrophic support contributes to corneal disease. Information regarding the relationship between nerve loss and effects on the corneal epithelium is limited. We investigated changes in the corneal epithelium and nerve morphology using three-dimensional imaging in vivo and in situ in a streptozotocin-induced diabetic mouse model. Streptozotocin-treated mice showed increased levels of serum glucose and growth retardation consistent with a severe diabetic state. A reduction in the length of the subbasal nerve plexus was evident after 6 weeks of disease. Loss of the subbasal nerve plexus was associated with corneal epithelial thinning and a reduction in basal epithelial cell density. In contrast, loss of the terminal epithelial nerves was associated with animal age. Importantly, this is the first rodent model of type 1 diabetes that shows characteristics of corneal epithelial thinning and a reduction in basal epithelial cell density, both previously have been documented in humans with diabetic corneal neuropathy. These findings indicate that in type 1 diabetes, nerve fiber damage is evident in the subbasal nerve plexus before terminal epithelial nerve loss and that neurotrophic support from both the subbasal nerve plexus and terminal epithelial nerves is essential for the maintenance of corneal epithelial homeostasis. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  2. Role of phospholipase A₂ (PLA₂) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis.

    Science.gov (United States)

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2013-08-01

    The aim of this study is to determine if the mannose-induced protein (MIP-133) from Acanthamoeba castellanii trophozoites induces apoptosis of corneal epithelial cells through a cytosolic phospholipase A2α (cPLA2α)-mediated pathway. The efficacy of cPLA2α inhibitors to provide protection against Acanthamoeba keratitis was examined in vivo. Chinese hamster corneal epithelial (HCORN) cells were incubated with or without MIP-133. MIP-133 induces significant increase in cPLA2α and macrophage inflammatory protein-2 (MIP-2/CXCL2) levels from corneal cells. Moreover, cPLA2α inhibitors, MAFP (Methyl-arachidonyl fluorophosphonate) and AACOCF3 (Arachidonyl trifluoromethyl ketone), significantly reduce cPLA2α and CXCL2 from these cells (P keratitis as compared with control animals. Collectively, the results indicate that cPLA2α is involved in MIP-133 induced apoptosis of corneal epithelial cells, polymorphonuclear neutrophil infiltration, and production of CXCL2. Moreover, cPLA2α inhibitors can be used as a therapeutic target in Acanthamoeba keratitis. Copyright © 2013. Published by Elsevier Ltd.

  3. 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.

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

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    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.

  5. [Inhibitory effect of diclofenac sodium on the proliferation of rabbit corneal epithelial cells in vitro].

    Science.gov (United States)

    Wu, Ningling; Du, Zhiyu

    2010-11-01

    To investigate the inhibitory effect of diclofenac sodium on rabbit corneal epithelial cells (RCECs) in vitro and explore its pharmacological mechanism. The fresh rabbit cornea was cultured to obtain the primary RCECs, and RCECs of passage 2 were used in this research. The cells were divided into experimental groups, the cells in which were incubated with different concentrations (18.18, 27.27, 36.36, 45.45, 54.55 μg/ml) of diclofenac sodium, and control group. The effect of diclofenac sodium on the proliferation of cells was measured by methyl thiazolyl thiazolium (MTT) assay 24, 48 and 72 h after incubation. While the RCECs were divided into experimental groups, the cells in which were incubated with 9 and 12.5 μg/ml diclofenac sodium, and control group. The cell cycle and apoptotic rate were observed by flow cytometer. MTT assay showed that diclofenac sodium had obvious inhibitory effect on RCECs, and the inhibition rate was increasing along with the increasing concentration of diclofenac sodium and the incubation time(Pdiclofenac sodium, the cells in G0/G1 phase were obviously increased, and the apoptosis cusp and apoptotic rate were increased. Diclofenac sodium exerts significant inhibitory effect on RCECs in a dosage-dependent manner, and it may function by inducing cell apoptosis and ceasing cell cycles.

  6. Corneal epithelial cell viability of an ex vivo porcine eye model.

    Science.gov (United States)

    Chan, Ka Yin; Cho, Pauline; Boost, Maureen

    2014-07-01

    The aim was to assess the consistency of corneal epithelial cell viability of an ex vivo porcine eye model. Six porcine eye models (four test and two control) were prepared for each experiment. The model has a computer-controlled mechanical arm, which could move the eyelid of the porcine eye and apply phosphate buffered saline to simulate blinking and lacrimation. The four test eyes were set up to simulate evaporative dry eyes with simulated lacrimation and blinking (one blink and one drop of buffered saline per minute) over three hours. Control A models were set up to collect pre-experimental baseline data, while those of control B were the same as the test eyes but without lacrimation and blinking simulation. All porcine eyes were kept in a closed chamber with temperature and humidity well controlled. After three hours, the cells of all eyes (except control A, which were assessed immediately before commencement of the experiment) were assessed. The eyes were first dipped into 0.4 per cent trypan blue solution. Following the dissection and separation of the cells, the number of dead cells were then counted under the microscope with a field size of 0.25 mm(2). The experiment was repeated 11 times. No significant differences were found in the number of dead cells among the four test eyes in both the central and peripheral cornea. There were significantly more dead cells in the test eyes compared to control A but significantly less when compared to control B. More dead cells were found in the central cornea than the peripheral cornea in the test eyes but the difference was not observed in controls A and B. Epithelial cell viabilities among the four porcine eye models with simulated lacrimation and blinking were consistent. The majority of cells were viable before the experiment and simulated lacrimation and blinking maintained more viable cells over time. © 2014 The Authors. Clinical and Experimental Optometry © 2014 Optometrists Association Australia.

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

    OpenAIRE

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

    2016-01-01

    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 ...

  8. Communication between corneal epithelial cells and trigeminal neurons is facilitated by purinergic (P2 and glutamatergic receptors.

    Directory of Open Access Journals (Sweden)

    Duane J Oswald

    Full Text Available Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2 receptors resulting in mobilization of a Ca(2+ wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+ wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+ mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+ mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+ waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.

  9. Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

    Science.gov (United States)

    Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla

    2016-02-01

    To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

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

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    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.

  11. Diclofenac protects cultured human corneal epithelial cells against hyperosmolarity and ameliorates corneal surface damage in a rat model of dry eye.

    Science.gov (United States)

    Sawazaki, Ryoichi; Ishihara, Tomoaki; Usui, Shinya; Hayashi, Erika; Tahara, Kayoko; Hoshino, Tatsuya; Higuchi, Akihiro; Nakamura, Shigeru; Tsubota, Kazuo; Mizushima, Tohru

    2014-04-21

    Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis. Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid. Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.

  12. 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.

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

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    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.

  14. Protective Effects of L-Carnitine Against Oxidative Injury by Hyperosmolarity in Human Corneal Epithelial Cells.

    Science.gov (United States)

    Hua, Xia; Deng, Ruzhi; Li, Jin; Chi, Wei; Su, Zhitao; Lin, Jing; Pflugfelder, Stephen C; Li, De-Quan

    2015-08-01

    L-carnitine suppresses inflammatory responses in human corneal epithelial cells (HCECs) exposed to hyperosmotic stress. In this study, we determined if L-carnitine induces this protective effect through suppression of reactive oxygen species (ROS)-induced oxidative damage in HCECs. Primary HCECs were established from donor limbal explants. A hyperosmolarity dry-eye model was used in which HCECs are cultured in 450 mOsM medium with or without L-carnitine for up to 48 hours. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and antioxidative enzymes were analyzed by 2',7'-dichlorofluorescein diacetate (DCFDA) kit, semiquantitative PCR, immunofluorescence, and/or Western blotting. Reactive oxygen species production increased in HCECs upon substitution of the isotonic medium with the hypertonic medium. L-carnitine supplementation partially suppressed this response. Hyperosmolarity increased cytotoxic membrane lipid peroxidation levels; namely, malondialdehyde (MDA) and hydroxynonenal (HNE), as well as mitochondria DNA release along with an increase in 8-OHdG and aconitase-2. Interestingly, these oxidative markers were significantly decreased by coculture with L-carnitine. Hyperosmotic stress also increased the mRNA expression and/or protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but inhibited the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), glutathione peroxidase-1 (GPX1), and peroxiredoxin-4 (PRDX4). However, L-carnitine partially reversed this altered imbalance between oxygenases and antioxidant enzymes induced by hyperosmolarity. Our findings demonstrate for the first time that L-carnitine protects HCECs from oxidative stress by lessening the declines in antioxidant enzymes and suppressing ROS production. Such suppression reduces membrane lipid oxidative damage markers and mitochondrial DNA damage.

  15. 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.

  16. Apoptosis of Corneal Epithelial Cells Caused by Ultraviolet B-induced Loss of K+ is Inhibited by Ba2+

    Science.gov (United States)

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

    2017-01-01

    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 Ba2+ blocks several K+ channels, we tested whether Ba2+-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/cm2. 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 Ba2+. K+ currents were activated in HCLE cells following UVB-exposure. These currents were reversibly blocked by 5 mM Ba2+. When HCLE cells were incubated with 5 mM Ba2+ 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 Ba2+ inhibits UVB-induced apoptosis by preventing loss of intracellular K+ when K+ channels are activated. Ba2+ 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. PMID:27189864

  17. 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.

  18. Myogel supports the ex-vivo amplification of corneal epithelial cells.

    Science.gov (United States)

    Francis, D; Abberton, K; Thompson, E; Daniell, M

    2009-03-01

    Limbal stem cell deficiency leads to conjunctivalisation of the cornea and subsequent loss of vision. The recent development of transplantation of ex-vivo amplified corneal epithelium, derived from limbal stem cells, has shown promise in treating this challenging condition. The purpose of this research was to compare a variety of cell sheet carriers for their suitability in creating a confluent corneal epithelium from amplified limbal stem cells. Cadaveric donor limbal cells were cultured using an explant technique, free of 3T3 feeder cells, on a variety of cell sheet carriers, including denuded amniotic membrane, Matrigel, Myogel and stromal extract. Comparisons in rate of growth and degree of differentiation were made, using immunocytochemistry (CK3, CK19 and ABCG2). The most rapid growth was observed on Myogel and denuded amniotic membrane, these two cell carriers also provided the most reliable substrata for achieving confluence. The putative limbal stem cell marker, ABCG2, stained positively on cells grown over Myogel and Matrigel but not for those propagated on denuded amniotic membrane. In the clinical setting amniotic membrane has been demonstrated to provide a suitable carrier for limbal stem cells and the resultant epithelium has been shown to be successful in treating limbal stem cell deficiency. Myogel may provide an alternative cell carrier with a further reduction in risk as it is has the potential to be derived from an autologous muscle biopsy in the clinical setting.

  19. 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.

  20. A rapid separation of two distinct populations of mouse corneal epithelial cells with limbal stem cell characteristics by centrifugation on percoll gradient

    Czech Academy of Sciences Publication Activity Database

    Krulová, Magdalena; Pokorná, Kateřina; Lenčová, Anna; Frič, Jan; Zajícová, Alena; Filipec, Martin; Forrester, J. V.; Holáň, Vladimír

    2008-01-01

    Roč. 49, č. 9 (2008), s. 3903-3908 ISSN 0146-0404 R&D Projects: GA AV ČR KAN200520804; GA MŠk 1M0506; GA ČR GD310/08/H077 Institutional research plan: CEZ:AV0Z50520514 Keywords : limbal stem cells * Percoll gradient * corneal epithelial cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.582, year: 2008

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

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    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

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

    International Nuclear Information System (INIS)

    Pan, Hong; Wu, Xinyi

    2012-01-01

    Highlights: ► Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-β. ► Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. ► Hypoxia inhibits Acanthamoeba-induced the activation of NF-κB and ERK1/2 in HCECs. ► Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. ► 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-β (IFN-β) 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-κB) 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-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB 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) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion

  3. Effects of Moraxella (Branhamella) ovis culture filtrates on bovine erythrocytes, peripheral mononuclear cells, and corneal epithelial cells.

    Science.gov (United States)

    Cerny, Henry E; Rogers, Douglas G; Gray, Jeffrey T; Smith, David R; Hinkley, Susanne

    2006-03-01

    Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease that affects cattle of all ages and that occurs worldwide. Piliated hemolytic Moraxella bovis is recognized as the etiologic agent of IBK. According to data from the Nebraska Veterinary Diagnostic Laboratory System, however, Moraxella (Branhamella) ovis has been isolated with increasing frequency from cattle affected with IBK. The objective of this study was, therefore, to examine M. ovis field isolates for the presence of the putative virulence factors of M. bovis. Culture filtrates from selected M. ovis field isolates demonstrated hemolytic activity on bovine erythrocytes and cytotoxic activity on bovine peripheral blood mononuclear cells and corneal epithelial cells. The hemolytic activity of the culture filtrates was attenuated after heat treatment. Polyclonal antibodies raised against the M. bovis hemolysin-cytotoxin also recognized a protein of approximately 98 kDa in a Western blot assay. These data indicate that the M. ovis field isolates examined produce one or more heat-labile exotoxins and may suggest that M. ovis plays a role in the pathogenesis of IBK.

  4. Tualang Honey Improves Human Corneal Epithelial Progenitor Cell Migration and Cellular Resistance to Oxidative Stress In Vitro

    Science.gov (United States)

    Tan, Jun Jie; Azmi, Siti Maisura; Yong, Yoke Keong; Cheah, Hong Leong; Lim, Vuanghao; Sandai, Doblin; Shaharuddin, Bakiah

    2014-01-01

    Stem cells with enhanced resistance to oxidative stress after in vitro expansion have been shown to have improved engraftment and regenerative capacities. Such cells can be generated by preconditioning them with exposure to an antioxidant. In this study we evaluated the effects of Tualang honey (TH), an antioxidant-containing honey, on human corneal epithelial progenitor (HCEP) cells in culture. Cytotoxicity, gene expression, migration, and cellular resistance to oxidative stress were evaluated. Immunofluorescence staining revealed that HCEP cells were holoclonal and expressed epithelial stem cell marker p63 without corneal cytokeratin 3. Cell viability remained unchanged after cells were cultured with 0.004, 0.04, and 0.4% TH in the medium, but it was significantly reduced when the concentration was increased to 3.33%. Cell migration, tested using scratch migration assay, was significantly enhanced when cells were cultured with TH at 0.04% and 0.4%. We also found that TH has hydrogen peroxide (H2O2) scavenging ability, although a trace level of H2O2 was detected in the honey in its native form. Preconditioning HCEP cells with 0.4% TH for 48 h showed better survival following H2O2-induced oxidative stress at 50 µM than untreated group, with a significantly lower number of dead cells (15.3±0.4%) were observed compared to the untreated population (20.5±0.9%, p<0.01). Both TH and ascorbic acid improved HCEP viability following induction of 100 µM H2O2, but the benefit was greater with TH treatment than with ascorbic acid. However, no significant advantage was demonstrated using 5-hydroxymethyl-2-furancarboxaldehyde, a compound that was found abundant in TH using GC/MS analysis. This suggests that the cellular anti-oxidative capacity in HCEP cells was augmented by native TH and was attributed to its antioxidant properties. In conclusion, TH possesses antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in HCEP cells in

  5. Role of phospholipase A2 (PLA2) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis

    Science.gov (United States)

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2013-01-01

    The aim of this study is to determine if the mannose-induced protein (MIP-133) from Acanthamoeba castellanii trophozoites induces apoptosis of corneal epithelial cells through a cytosolic phospholipase A2α (cPLA2α)-mediated pathway. The efficacy of cPLA2α inhibitors to provide protection against Acanthamoeba keratitis was examined in vivo. Chinese hamster corneal epithelial (HCORN) cells were incubated with or without MIP-133. MIP-133 induces significant increase in cPLA2α and macrophage inflammatory protein-2 (MIP-2/CXCL2) levels from corneal cells. Moreover, cPLA2α inhibitors, MAFP (Methyl-arachidonyl fluorophosphonate) and AACOCF3 (Arachidonyl trifluoromethyl ketone), significantly reduce cPLA2α and CXCL2 from these cells (Pkeratitis as compared with control animals. Collectively, the results indicate that cPLA2α is involved in MIP-133 induced apoptosis of corneal epithelial cells, polymorphonuclear neutrophil infiltration, and production of CXCL2. Moreover, cPLA2α inhibitors can be used as a therapeutic target in Acanthamoeba keratitis. PMID:23792108

  6. 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

  7. Development of Acyclovir-Loaded Albumin Nanoparticles and Improvement of Acyclovir Permeation Across Human Corneal Epithelial T Cells.

    Science.gov (United States)

    Suwannoi, Panita; Chomnawang, Mullika; Sarisuta, Narong; Reichl, Stephan; Müller-Goymann, Christel C

    2017-12-01

    The aim of the present study was to develop acyclovir (ACV) ocular drug delivery systems of bovine serum albumin (BSA) nanoparticles as well as to assess their in vitro transcorneal permeation across human corneal epithelial (HCE-T) cell multilayers. The ACV-loaded BSA nanoparticles were prepared by desolvation method along with physicochemical characterization, cytotoxicity, as well as in vitro transcorneal permeation studies across HCE-T cell multilayers. The nanoparticles appeared to be spherical in shape and nearly uniform in size of about 200 nm. The size of nanoparticles became smaller with decreasing BSA concentration, while the ratios of water to ethanol seemed not to affect the size. Increasing the amount of ethanol in desolvation process led to significant reduction of drug entrapment of nanoparticles with smaller size and more uniformity. The ACV-loaded BSA nanoparticles prepared were shown to have no cytotoxic effect on HCE-T cells used in permeation studies. The in vitro transcorneal permeation results revealed that ACV could permeate through the HCE-T cell multilayers significantly higher from BSA nanoparticles than from aqueous ACV solutions. The ACV-loaded BSA nanoparticles could be prepared by desolvation method without glutaraldehyde in the formulation. ACV could increasingly permeate through the multilayers of HCE-T cells from the ACV-loaded BSA nanoparticles. Therefore, the ACV-loaded BSA nanoparticles could be a highly potential ocular drug delivery system.

  8. TNF-R1 and FADD mediate UVB-Induced activation of K+ channels in corneal epithelial cells

    Science.gov (United States)

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

    2017-01-01

    The goal of this study was to elucidate the role of Fas, TNF-R1, FADD and cytochrome c in UVB-induced K+ channel activation, an early step in UVB-induced apoptosis, in human corneal limbal epithelial (HCLE) cells. HCLE cells were treated with Fas, TNF-R1 or FADD siRNA and exposed to 80 or 150 mJ/cm2 UVB. K+ channel activation and loss of intracellular K+ were measured using whole-cell patch-clamp recording and ion chromatography, respectively. Cytochrome c was measured with an ELISA kit. Cells in which Fas was knocked down exhibited identical UVB-induced K+ channel activation and loss of intracellular K+ to control cells. Cells in which TNF-R1 or FADD were knocked down demonstrated reduced K+ channel activation and decreased loss of intracellular K+ following UVB, relative to control cells. Application of TNF-α, the natural ligand of TNF-R1, to HCLE cells induced K+ channel activation and loss of intracellular K+. Cytochrome c was translocated to the cytosol by 2 h after exposure to 150 mJ/cm2 UVB. However, there was no release by 10 min post-UVB. The data suggest that UVB activates TNF-R1, which in turn may activate K+ channels via FADD. This conclusion is supported by the observation that TNF-α also causes loss of intracellular K+. This signaling pathway appears to be integral to UVB-induced K+ efflux, since knockdown of TNF-R1 or FADD inhibits the UVB-induced K+ efflux. The lack of rapid cytochrome c translocation indicates cytochrome c does not play a role in UVB-induced K+ channel activation. PMID:27818316

  9. Effects of Moraxella (Branhamella) ovis Culture Filtrates on Bovine Erythrocytes, Peripheral Mononuclear Cells, and Corneal Epithelial Cells†

    Science.gov (United States)

    Cerny, Henry E.; Rogers, Douglas G.; Gray, Jeffrey T.; Smith, David R.; Hinkley, Susanne

    2006-01-01

    Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease that affects cattle of all ages and that occurs worldwide. Piliated hemolytic Moraxella bovis is recognized as the etiologic agent of IBK. According to data from the Nebraska Veterinary Diagnostic Laboratory System, however, Moraxella (Branhamella) ovis has been isolated with increasing frequency from cattle affected with IBK. The objective of this study was, therefore, to examine M. ovis field isolates for the presence of the putative virulence factors of M. bovis. Culture filtrates from selected M. ovis field isolates demonstrated hemolytic activity on bovine erythrocytes and cytotoxic activity on bovine peripheral blood mononuclear cells and corneal epithelial cells. The hemolytic activity of the culture filtrates was attenuated after heat treatment. Polyclonal antibodies raised against the M. bovis hemolysin-cytotoxin also recognized a protein of approximately 98 kDa in a Western blot assay. These data indicate that the M. ovis field isolates examined produce one or more heat-labile exotoxins and may suggest that M. ovis plays a role in the pathogenesis of IBK. PMID:16517853

  10. Corneal epithelial inclusion cyst in a dog

    Directory of Open Access Journals (Sweden)

    Campos Carla de Freitas

    2002-01-01

    Full Text Available An unilateral corneal epithelial inclusion cyst in a 7-year-old male Boxer dog is reported. The cyst had been observed for thirty days, was unique, not congenital and only one eye was involved. Seven months prior to the referral the dog had manifested indolent corneal ulcer treated with grade keratotomy and third eyelid flap. The cyst was removed by superficial keratectomy followed by a conjunctival pedicle graft. Recovery was uncomplicated and there wasn?t recurrence seven months after the surgery.

  11. Corneal collagen crosslinking for keratoconus or corneal ectasia without epithelial debridement.

    Science.gov (United States)

    Hirji, N; Sykakis, E; Lam, F C; Petrarca, R; Hamada, S; Lake, D

    2015-06-01

    Corneal collagen crosslinking (CXL) is a relatively new technique to reduce the progression of keratoconus. The technique can be performed with or without complete debridement of the corneal epithelium. We describe a novel intermediate technique involving mechanical disruption of the epithelium, and evaluate its safety and efficacy. The case notes of 128 eyes with progressive keratoconus or iatrogenic corneal ectasia who had undergone CXL using the epithelial disruption technique were retrospectively reviewed. Thin corneas were treated with hypotonic riboflavin. All others were treated with an isotonic solution. Note was made of preoperative and postoperative parameters, including uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), refraction, endothelial cell count, and corneal tomography. Occurrence of procedure-related complications was recorded. Statistical analyses were performed using the paired sample t-test and Wilcoxon signed-rank test, with a level of Pcorneal ectasia, and may be better tolerated by patients than the epithelium-off technique.

  12. Advanced glycation end products delay corneal epithelial wound healing through reactive oxygen species generation.

    Science.gov (United States)

    Shi, Long; Chen, Hongmei; Yu, Xiaoming; Wu, Xinyi

    2013-11-01

    Delayed healing of corneal epithelial wounds is a serious complication in diabetes. Advanced glycation end products (AGEs) are intimately associated with the diabetic complications and are deleterious to the wound healing process. However, the effect of AGEs on corneal epithelial wound healing has not yet been evaluated. In the present study, we investigated the effect of AGE-modified bovine serum albumin (BSA) on corneal epithelial wound healing and its underlying mechanisms. Our data showed that AGE-BSA significantly increased the generation of intracellular ROS in telomerase-immortalized human corneal epithelial cells. However, the generation of intracellular ROS was completely inhibited by antioxidant N-acetylcysteine (NAC), anti-receptor of AGEs (RAGE) antibodies, or the inhibitor of NADPH oxidase. Moreover, AGE-BSA increased NADPH oxidase activity and protein expression of NADPH oxidase subunits, p22phox and Nox4, but anti-RAGE antibodies eliminated these effects. Furthermore, prevention of intracellular ROS generation using NAC or anti-RAGE antibodies rescued AGE-BSA-delayed epithelial wound healing in porcine corneal organ culture. In conclusion, our results demonstrated that AGE-BSA impaired corneal epithelial wound healing ex vivo. AGE-BSA increased intracellular ROS generation through NADPH oxidase activation, which accounted for the delayed corneal epithelial wound healing. These results may provide better insights for understanding the mechanism of delayed healing of corneal epithelial wounds in diabetes.

  13. Nano-sized iron particles may induce multiple pathways of cell death following generation of mistranscripted RNA in human corneal epithelial cells.

    Science.gov (United States)

    Park, Eun-Jung; Chae, Jae-Byoung; Kang, Seuyoung; Lyu, Jungmook; Jeong, Uiseok; Yeom, Changjoo; Kim, Younghun; Chang, Jaerak

    2017-08-01

    Iron is closely associated with an ambient particulate matters-induced inflammatory response, and the cornea that covers the front of the eye, is among tissues exposed directly to ambient particulate matters. Prior to this study, we confirmed that nano-sized iron particles (FeNPs) can penetrate the cornea. Thus, we identified the toxic mechanism of FeNPs using human corneal epithelial cells. At 24h after exposure, FeNPs located inside autophagosome-like vacuoles or freely within human corneal epithelial cells. Level of inflammatory mediators including nitric oxide, cytokines, and a chemokine was notably elevated accompanied by the increased generation of reactive oxygen species. Additionally, cell proliferation dose-dependently decreased, and level of multiple pathways of cell death-related indicators was clearly altered following exposure to FeNPs. Furthermore, expression of gene encoding DNA binding protein inhibitor (1, 2, and 3), which are correlated to inhibition of the binding of mistranscripted RNA, was significantly down-regulated. More importantly, expression of p-Akt and caspase-3 and conversion to LC3B-II from LC3B-I was enhanced by pretreatment with a caspase-1 inhibitor. Taken together, we suggest that FeNPs may induce multiple pathways of cell death via generation of mistranscripted RNA, and these cell death pathways may influence by cross-talk. Furthermore, we propose the need of further study for the possibility of tumorigenesis following exposure to FeNPs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Involvement of the Extrinsic and Intrinsic Pathways in Ultraviolet B-Induced Apoptosis of Corneal Epithelial Cells

    Science.gov (United States)

    Ubels, John L.; Glupker, Courtney D.; Schotanus, Mark P.; Haarsma, Loren D.

    2015-01-01

    The goal of this study was to elucidate the pathway by which UVB initiates efflux of K+ and subsequently apoptosis in human corneal limbal epithelial (HCLE) cells. The initial focus of the study was on the extrinsic pathway involving Fas. HCLE cells transfected with Fas siRNA were exposed to 80–150 mJ/cm2 UVB and incubated in culture medium with 5.5 mM K+. Knock down of Fas resulted in limited reduction in UVB-induced caspase-8 and -3 activity. Patch-clamp recordings showed no difference in UVB-induced normalized K+ currents between Fas transfected and control cells. Knockdown of caspase-8 had no effect on the activation of caspase-3 following UVB exposure, while a caspase-8 inhibitor completely eliminated UVB activation of caspase-3. This suggests that caspase-8 is a robust enzyme, able to activate caspase-3 via residual caspase-8 present after knockdown, and that caspase-8 is directly involved in the UVB activation of caspase-3. Inhibition of caspase-9 significantly decreased the activation of caspases-8 and -3 in response to UVB. Knockdown of Apaf-1, required for activation of caspase-9, resulted in a significant reduction in UVB-induced activation of caspases-9, -8, and -3. Knockdown of Apaf-1 also inhibited intrinsic and UVB-induced levels of apoptosis, as determined by DNA fragmentation measured by TUNEL assay. In UVB exposed cultures treated with caspase-3 inhibitor, the percentage of apoptotic cells was reduced to control levels, confirming the necessity of caspase-3 activation in DNA fragmentation. The lack of effect of Fas knockdown on K+ channel activation, as well as the limited effect on activation of caspases-8 and -3, strongly suggest that Fas and the extrinsic pathway is not of primary importance in the initiation of apoptosis in response to UVB in HCLE cells. Inhibition of caspase-8 and -3 activation following inhibition of caspase-9, as well as reduction in activation of caspases-9, -8, and -3 and DNA fragmentation in response to Apaf-1

  15. [Corneal epithelial changes of soft contact lens wearers under a transmission electron microscope].

    Science.gov (United States)

    Gu, Hao; Tang, Li

    2015-03-01

    To investigate the difference in corneal epithelium between patients with and without soft contact lens (SCL) wearing, and to analyze corneal epithelial changes of the eyes with long-time SCL wearing. In this cross sectional study, the subjects were divided into two groups: 13 patients of daily SCL wearers and 11 control subjects who had never worn contact lenses. The flap of corneal epithelium was observed by transmission electron microscopy. The corneal epithelial microvillus density was compared between the two groups. Transmission electron micrographs of the control group showed a tight connection between cells, regularly aligned basal cells, and continuous basement membrane. Compared with the control group, SCL wearers showed incomplete basement membrane, swollen epithelial cells, swollen mitochondria, and widened intercellular interstices. The density of corneal epithelium microvilli [(0.071466 +/- 0.015889)/microm2 vs. (0.139851 +/- 0.024171)/micro2] was lower (t = 8.312, P < 0.05). Long-term SCL wearing can induce remarkable changes of corneal epithelial tissue, and the density of corneal epithelial microvilli decreases.

  16. 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

  17. 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

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

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    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.

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

    Science.gov (United States)

    Zhang, Ting; Wang, Yuexin; Jia, Yanni; Liu, Dongle; Li, Suxia; Shi, Weiyun; Gao, Hua

    2016-01-01

    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.

  20. Effects of Lutein on Hyperosmoticity-Induced Upregulation of IL-6 in Cultured Corneal Epithelial Cells and Its Relevant Signal Pathways

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    Shih-Chun Chao

    2016-01-01

    Full Text Available Dry eye is a common disorder characterized by deficiency of tear. Hyperosmoticity of tear stimulates inflammation and damage of ocular surface tissues and plays an essential role in the pathogenesis of dry eye. Cultured human corneal epithelial (CE cells were used for the study of effects of lutein and hyperosmoticity on the secretion of IL-6 by CE cells. Cell viability of CE cells was not affected by lutein at 1–10 μM as determined by MTT assay. Hyperosmoticity significantly elevated the secretion of IL-6 by CE cells as measured by ELISA analysis. The constitutive secretion of IL-6 was not affected by lutein. Lutein significantly and dose-dependently inhibited hyperosmoticity-induced secretion of IL-6. Phosphorylated- (p- p38 MAPK, p-JNK levels in cell lysates and NF-κB levels in cell nuclear extracts were increased by being exposed to hyperosmotic medium. JNK, p38, and NF-κB inhibitors decreased hyperosmoticity-induced secretion of IL-6. Lutein significantly inhibited hyperosmoticity-induced elevation of NF-κB, p38, and p-JNK levels. We demonstrated that lutein inhibited hyperosmoticity-induced secretion of IL-6 in CE cells through the deactivation of p38, JNK, and NF-κB pathways. Lutein may be a promising agent to be explored for the treatment of dry eye.

  1. Effect of Lipoglycans from Mycobacterium Chelonae on the expression of inflammatory factors IL-8 and IL-6 in human corneal epithelial cells and its possible signal transduction pathway

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    Chun-Zhou Tang

    2015-06-01

    Full Text Available AIM: To study the influence of Lipoglycans from Mycobacterium Chelonae(Cheon the expression of IL-6 and IL-8 in human corneal epithelia cells and its possible signal transduction pathway.METHODS: Lipoglycans was extracted by the Triton X-114 phase partitioning. Lipoglycans from Che were purified, by successive detergent and phenol extractions. Lipoglycans were separated by gel filtration on a Sephacryl 200 column and Sephacryl 100 column in series, followed by extensive dialisis. Purified Lipoglycans(50μg/mLwere added into culture medium to stimulate primary human corneal epithelial(HCEcells. Cells and supernatant were collected at 0, 6, 12, 24h after the stimulation. The IL-6 and IL-8 expression at mRNA level was assayed by using real time RT-PCR and the secreted IL-6 and IL-8 in the supernatants was measured by ELISA. Immunochemistry was used to detect the expression and location of NF-κB in HCE cells.RESULTS: After the treatment of Lipoglycans, the expression of IL-8 and IL-6 at mRNA level obviouly increased within 12h, and reached peak level at 6h(IL-8 was 36.8 times that of the blank control, and IL-6 was 32.7 times. Compared with the blank control group, the expression of IL-8 at protein level in the supernatant increased 2.8 folds at 6h(P>0.05, 13.4 folds at 12h(PPPPPCONCLUSION: Lipoglycans from Che can induce HCE cells to produce inflammatory factors(IL-6 and IL-8, and its signal transduction pathway probably is mediated by NF-κB.

  2. The Use of an IL-1 Receptor Antagonist Peptide to Control Inflammation in the Treatment of Corneal Limbal Epithelial Stem Cell Deficiency

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    E. Fok

    2015-01-01

    Full Text Available Corneal limbal stem cell deficiency (LSCD may be treated using ex vivo limbal epithelial stem cells (LESCs derived from cadaveric donor tissue. However, continuing challenges exist around tissue availability, inflammation, and transplant rejection. Lipopolysaccharide (LPS or recombinant human IL-1β stimulated primary human keratocyte and LESC models were used to investigate the anti-inflammatory properties of a short chain, IL-1 receptor antagonist peptide for use in LESC sheet growth to control inflammation. The peptide was characterized using mass spectroscopy and high performance liquid chromatography. Peptide cytotoxicity, patterns of cell cytokine expression in response to LPS or IL-1β stimulation, and peptide suppression of this response were investigated by MTS/LDH assays, ELISA, and q-PCR. Cell differences in LPS stimulated toll-like receptor 4 expression were investigated using immunocytochemistry. A significant reduction in rIL-1β stimulated inflammatory cytokine production occurred following LESC and keratocyte incubation with anti-inflammatory peptide and in LPS stimulated IL-6 and IL-8 production following keratocyte incubation with peptide (1 mg/mL P<0.05. LESCs produced no cytokine response to LPS stimulation and showed no TLR4 expression. The peptide supported LESC growth when adhered to a silicone hydrogel contact lens indicating potential use in improved LESC grafting through suppression of inflammation.

  3. Application of SV40 T-transformed human corneal epithelial cells to evaluate potential irritant chemicals for in vitro alternative eye toxicity.

    Science.gov (United States)

    Kim, Cho-Won; Park, Geon-Tae; Bae, Ok-Nam; Noh, Minsoo; Choi, Kyung-Chul

    2016-01-01

    Assessment of eye irritation potential is important to human safety, and it is necessary for various cosmetics and chemicals that may contact the human eye. Until recently, the Draize test was considered the standard method for estimating eye irritation, despite its disadvantages such as the need to sacrifice many rabbits for subjective scoring. Thus, we investigated the cytotoxicity and inflammatory response to standard eye irritants using SV40 T-transformed human corneal epithelial (SHCE) cells as a step toward development of an animal-free alternative eye irritation test. MTT and NRU assays of cell viability were performed to investigate the optimal experimental conditions for SHCE cell viability when cells were exposed to sodium dodecyl sulfate (SDS) as a standard eye irritant at 6.25×10(-3) to 1×10(-1)%. Additionally, cell viability of SHCE cells was examined in response to six potential eye irritants, benzalkonium chloride, dimethyl sulfoxide, isopropanol, SDS, Triton X-100 and Tween 20 at 5×10(-3) to 1×10(-1)%. Finally, we estimated the secretion level of cytokines in response to stimulation by eye irritants in SHCE cells. SHCE cells showed a good response to potential eye irritants when the cells were exposed to potential irritants for 10min at room temperature (RT), and cytokine production increased in a concentration-dependent manner, indicating that cytotoxicity and cytokine secretion from SHCE cells may be well correlated with the concentrations of irritants. Taken together, these results suggest that SHCE cells could be an excellent alternative in vitro model to replace in vivo animal models for eye irritation tests. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Construction of tissue-engineered full-thickness cornea substitute using limbal epithelial cell-like and corneal endothelial cell-like cells derived from human embryonic stem cells.

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    Zhang, Canwei; Du, Liqun; Sun, Peng; Shen, Lin; Zhu, Jing; Pang, Kunpeng; Wu, Xinyi

    2017-04-01

    The aim of this study was to construct a full-thickness artificial cornea substitute in vitro by coculturing limbal epithelial cell-like (LEC-like) cells and corneal endothelial cell-like (CEC-like) cells derived from human embryonic stem cells (hESCs) on APCM scaffold. A 400 μm thickness, 11 mm diameter APCM lamella containing Bowman's membrane was prepared as the scaffold using trephine and a special apparatus made by ourselves. LEC-like cells and CEC-like cells, derived from hESCs as our previously described, were cocultured on the scaffold using a special insert of 24-well plates that enabled seeding both sides of the scaffold. Three or four layers of epithelium-like cells and a uniform monolayer of CEC-like cells could be observed by H&E staining. The thickness, endothelial cell density, and mechanical properties of the construct were similar to that of native rabbit corneas. Immunofluorescence analysis showed expression of ABCG2 and CK3 in the epithelium-like cell layers and expression of N-cadherin, ZO-1 and Na+/K + ATPase in the CEC-like cells. The corneal substitutes were well integrated within the host corneas, and the transparency increased gradually in 8-week follow-up after transplantation in the rabbits. These results suggest that the strategy we developed is feasible and effective for construction of tissue-engineered full-thickness cornea substitute with critical properties of native cornea. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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    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.

  6. Effect of Amniotic Membrane Suspension (AMS) and Amniotic Membrane Homogenate (AMH) on Human Corneal Epithelial Cell Viability, Migration and Proliferation In Vitro.

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    Wu, Ming-Feng; Stachon, Tanja; Langenbucher, Achim; Seitz, Berthold; Szentmáry, Nóra

    2017-03-01

    To analyze the effects of different concentrations of amniotic membrane suspension (AMS) or amniotic membrane homogenate (AMH) on human corneal epithelial cell (HCEC) viability, migration and proliferation. Amniotic membranes (AMs) of 13 placentas were prepared and stored at -80°C. For AMS preparation, following de-freezing, AM pieces were inserted in six-well plates and 5 ml Dulbecco's Modified Eagle's Medium (DMEM)/F12 (with 5% fetal bovine serum, FBS) per gram tissue was added for 96 h. After removal of the AM, the remaining supernatant was collected for experiments. For AMH preparation, following de-freezing, AMs were homogenized in liquid nitrogen and 5 ml DMEM/F12 (with 5% FBS) per gram tissue was added. Following centrifugation, the supernatant was collected for experiments. HCECs were expanded and incubated in DMEM/F12, 5% FBS supplemented by 15%, 30% or 100% AMS or 15% or 30% AMH. Viability was analyzed using Cell Proliferation Kit XTT, migration using wound healing assay and proliferation by the cell proliferation ELISA BrdU kit. HCEC viability remained unchanged using 15% or 30% AMS (p = 1.0 for both); however, it decreased significantly using 100% AMS (p < 0.001) or 15% (p = 0.041) or 30% AMH (p < 0.001), compared to controls. Using 15% or 30% AMS, HCEC migration increased significantly (p < 0.001 for both). Using 15% or 30% AMH (p = 0.153; p = 0.083), HCEC migration remained unchanged and 100% AMS inhibited HCEC migration (p < 0.001). Next, 15% and 30% AMS had no effect on HCEC proliferation (p = 0.454 and p = 0.119), but 100% AMS (p < 0.001) and 15% (p = 0.002) and 30% AMH (p = 0.001) inhibited HCEC proliferation significantly. With unchanged HCEC viability and proliferation and increased HCEC migration, 15% and 30% AMS application seems to be the most appropriate method to support epithelial healing.

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

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    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.

  8. 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.

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

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    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.

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

    OpenAIRE

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

    2011-01-01

    Wounding downregulates PTEN and activates the PI3 kinase/Akt pathway. Pharmacologic inhibition of PTEN stimulates the motility of corneal epithelial cells and corneal wound healing. These results imply that the inhibition of PTEN may be a plausible approach for corneal wounds.

  11. Adipose Derived Stem Cells for Corneal Wound Healing after Laser Induced Corneal Lesions in Mice

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    Marco Zeppieri

    2017-12-01

    Full Text Available The aim of our study was to assess the clinical effectiveness of topical adipose derived stem cell (ADSC treatment in laser induced corneal wounds in mice by comparing epithelial repair, inflammation, and histological analysis between treatment arms. Corneal lesions were performed on both eyes of 40 mice by laser induced photorefractive keratectomy. All eyes were treated with topical azythromycin bid for three days. Mice were divided in three treatment groups (n = 20, which included: control, stem cells and basic serum; which received topical treatment three times daily for five consecutive days. Biomicroscope assessments and digital imaging were performed by two masked graders at 30, 54, 78, 100, and 172 h to analyze extent of fluorescein positive epithelial defect, corneal inflammation, etc. Immunohistochemical techniques were used in fixed eyes to assess corneal repair markers Ki67, α Smooth Muscle Actin (α-SMA and E-Cadherin. The fluorescein positive corneal lesion areas were significantly smaller in the stem cells group on days 1 (p < 0.05, 2 (p < 0.02 and 3. The stem cell treated group had slightly better and faster re-epithelization than the serum treated group in the initial phases. Comparative histological data showed signs of earlier and better corneal repair in epithelium and stromal layers in stem cell treated eyes, which showed more epithelial layers and enhanced wound healing performance of Ki67, E-Cadherin, and α-SMA. Our study shows the potential clinical and histological advantages in the topical ADSC treatment for corneal lesions in mice.

  12. Modulation of inflammation and autophagy pathways by trehalose containing eye drop formulation in corneal epithelial cells: implications for dry eye disease

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    Trailokyanath Panigrahi

    2017-10-01

    Full Text Available Ocular surface inflammation is an immunological perturbation activated in response to various adverse conditions and is a key biomarker to understand the disease pathology and its underlying immunological landscape [1]. The molecular link between Inflammation and autophagy, often implicated in disease conditions, is poorly understood. The aim of this study is to understand the regulation of inflammation signaling pathways by using a well-established modulator of autophagy, trehalose (TRE, on desiccation stress-induced inflammation in SV40 immortalized human corneal epithelial cells. To mimic the dry eye condition, HCE cells were exposed to desiccation stress at 80% confluency in a six well tissue culture plate. The medium was completely aspirated and cells were kept for drying at room temperature for 10 min. Fresh medium with TRE was added and incubated for 6 hrs. The regulation of induced inflammatory and autophagic gene expression and protein activation by TRE formulation (1.2% was studied. Optimal drug treatment concentrations were determined by dose escalation cytotoxicity studies. Gene expression was evaluated by quantitative PCR, while protein expression and functions were tested by immunoblotting and fluorescence imaging (Cyto-ID, Lysotracker Red. TRE formulation was able to rescue the morphological changes due to desiccation stress. Live to dead cell ratio increased upon TRE treatment. TRE treatment reduced inflammation induced gene expression of IL-6 (2%, MCP-1 (33.31%, IL-8 (9.56%, MMP-9 (18.96%, and TNFα (58.16% in HCE. Active form of p38, p44/42, and p65 protein levels were altered significantly by TRE treatment. LAMP1 and LC3 autophagy protein markers were also altered with desiccation stress and TRE treatment. The data demonstrate that TRE formulation is effective in reducing desiccation stress induced inflammation in HCE. Further increased phosphorylation of p38, p44/42 and elevated levels of LC3 and LAMP1 suggest that induction

  13. Effects of L-carnitine, erythritol and betaine on pro-inflammatory markers in primary human corneal epithelial cells exposed to hyperosmotic stress.

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    Hua, Xia; Su, Zhitao; Deng, Ruzhi; Lin, Jing; Li, De-Quan; Pflugfelder, Stephen C

    2015-07-01

    To explore the effects of osmoprotectants on pro-inflammatory mediator production in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress. HCECs cultured in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media with or without prior incubation with 2-20 mM of l-carnitine, erythritol or betaine for different time periods. The mRNA expression and protein production of pro-inflammatory markers in HCECs were evaluated by RT-qPCR and ELISA. Hyperosmolar media significantly stimulated the mRNA and protein expression of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, and chemokines, IL-8, CCL2 and CCL20 in HCECs in an osmolarity dependent manner. The stimulated expression of these pro-inflammatory mediators was significantly but differentially suppressed by l-carnitine, erythritol or betaine. l-Carnitine displayed the greatest inhibitory effects and down-regulated 54-77% of the stimulated mRNA levels of TNF-α (down from 12.3-5.7 fold), IL-1β (2.2-0.9 fold), IL-6 (7.3-2.9 fold), IL-8 (4.6-2.0 fold), CCL2 (15.3-3.5 fold) and CCL20 (4.1-1.5 fold) in HCECs exposed to 450 mOsM. The stimulated protein production of TNF-α, IL-1β, IL-6 and IL-8 was also significantly suppressed by l-carnitine, erythritol and betaine. l-carnitine suppressed 49-79% of the stimulated protein levels of TNF-α (down from 81.3 to 17.4 pg/ml), IL-1β (56.9-29.2 pg/ml), IL-6 (12.8-4.6 ng/ml) and IL-8 (21.2-10.9 ng/ml) by HCECs exposed to 450 mOsM. Interestingly, hyperosmolarity stimulated increase in mRNA and protein levels of TNF-α, IL-1β and IL-6 were significantly suppressed by a transient receptor potential vanilloid channel type 1 (TRPV1) activation inhibitor capsazepine. l-carnitine, erythritol and betaine function as osmoprotectants to suppress inflammatory responses via TRPV1 pathway in HCECs exposed to hyperosmotic stress. Osmoprotectants may have efficacy in reducing innate inflammation in dry eye disease.

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

    OpenAIRE

    Zhang, Ting; Wang, Yuexin; Jia, Yanni; Liu, Dongle; Li, Suxia; Shi, Weiyun; Gao, Hua

    2016-01-01

    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...

  15. Temporary corneal stem cell dysfunction after radiation therapy

    International Nuclear Information System (INIS)

    Hiroshi, Fujishima; Kazuo, Tsubota

    1996-01-01

    Radiation therapy can cause corneal and conjuctival abnormalities that sometimes require surgical treatment. Corneal stem cell dysfunction is described, which recovered after the cessation of radiation. Methods - A 44-year-old man developed a corneal epithelial abnormality associated with conjuctival and corneal inflammation following radiation therapy for maxillary cancer. Examination of brush cytology samples showed goblet cells in the upper and lower parts of the cornea, which showed increased fluorescein permeability, and intraepithelial lymphocytes. Impression cytology showed goblet cells in the same part of the cornea. Specular microscopy revealed spindle type epithelial cells. Patient follow up included artificial tears and an antibiotic ophthalmic ointment. The corneal abnormalities resolved after 4 months with improved visual acuity without any surgical intervention, but the disappearance of the palisades of Vogt did not recover at 1 year after radiation. Radiation therapy in this patient caused temporary stem cell dysfunction which resulted in conjunctivalisation in a part of the cornea. Although limbal stem cell function did not fully recover, this rare case suggested that medical options should be considered before surgery. (Author)

  16. Epithelial basement membrane injury and regeneration modulates corneal fibrosis after pseudomonas corneal ulcers in rabbits.

    Science.gov (United States)

    Marino, Gustavo K; Santhiago, Marcony R; Santhanam, Abirami; Lassance, Luciana; Thangavadivel, Shanmugapriya; Medeiros, Carla S; Bose, Karthikeyan; Tam, Kwai Ping; Wilson, Steven E

    2017-08-01

    The purpose of this study was to investigate whether myofibroblast-related fibrosis (scarring) after microbial keratitis was modulated by the epithelial basement membrane (EBM) injury and regeneration. Rabbits were infected with Pseudomonas aeruginosa after epithelial scrape injury and the resultant severe keratitis was treated with topical tobramycin. Corneas were analyzed from one to four months after keratitis with slit lamp photos, immunohistochemistry for alpha-smooth muscle actin (α-SMA) and monocyte lineage marker CD11b, and transmission electron microscopy. At one month after keratitis, corneas had no detectible EBM lamina lucida or lamina densa, and the central stroma was packed with myofibroblasts that in some eyes extended to the posterior corneal surface with damage to Descemet's membrane and the endothelium. At one month, a nest of stromal cells in the midst of the SMA + myofibroblasts in the stroma that were CD11b+ may be fibrocyte precursors to myofibroblasts. At two to four months after keratitis, the EBM fully-regenerated and myofibroblasts disappeared from the anterior 60-90% of the stroma of all corneas, except for one four-month post-keratitis cornea where anterior myofibroblasts were still present in one localized pocket in the cornea. The organization of the stromal extracellular matrix also became less disorganized from two to four months after keratitis but remained abnormal compared to controls at the last time point. Myofibroblasts persisted in the posterior 10%-20% of posterior stroma even at four months after keratitis in the central cornea where Descemet's membrane and the endothelium were damaged. This study suggests that the EBM has a critical role in modulating myofibroblast development and fibrosis after keratitis-similar to the role of EBM in fibrosis after photorefractive keratectomy. Damage to EBM likely allows epithelium-derived transforming growth factor beta (TGFβ) to penetrate the stroma and drive development and

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

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    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.

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

    Science.gov (United States)

    de Araujo, Aline Lütz; Gomes, José Álvaro Pereira

    2015-06-26

    Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there are many challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.

  19. Alterations of epithelial adhesion molecules and basement membrane components in lattice corneal dystrophy (LCD).

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    Resch, Miklós D; Schlötzer-Schrehardt, Ursula; Hofmann-Rummelt, Carmen; Kruse, Friedrich E; Seitz, Berthold

    2009-08-01

    The aim of the study was to investigate the histopathological and ultrastructural correlate of delayed epithelial healing in eyes with lattice corneal dystrophy (LCD). Corneal buttons from 4 patients with LCD (two with subepithelial, two with stromal amyloid deposits) and 2 control corneas were examined. Cell-matrix adhesion molecules and basement membrane components of the corneal epithelium were analyzed by immunohistochemistry and hemidesmosomes between epithelium and stroma were quantified by transmission electron microscopy (TEM). By TEM well-developed hemidesmosomes anchored the basal epithelial cells to the underlying basement membrane in all normal and LCD corneas. Hemidesmosome density was not significantly different in subepithelial (224.7 +/- 34.1/100 microm) and stromal (234.3 +/- 36.3/100 microm) LCD compared to controls (241.3 +/- 26.8/100 microm). The basement membrane was interrupted in subepithelial, but continuous in stromal LCD. Integrin alpha6 and beta4 staining formed a continuous line along the basal surface of the corneal epithelium in control corneas, whereas it appeared discontinuous and patchy both in subepithelial and stromal forms of LCD. Staining for alphaV integrin showed irregular staining patterns, i.e. enhanced labelling intensity in subepithelial and interrupted pattern in stromal LCD, respectively. Integrins alpha3, beta1, beta2, and beta5, dystroglycan, and plectin were not markedly different in dystrophic corneas. Type VII collagen showed a discontinuous staining in subepithelial forms of LCD. In stromal forms of LCD, type VII collagen staining occurred in additional patches underneath the epithelial basement membrane zone. Type XVII collagen staining was reduced in subepithelial LCD. Laminin-1, laminin-5 and laminin gamma2 showed variable irregular staining patterns in dystrophic corneas with focal interruptions, focal thickenings, and reduplications of basement membrane. Some irregularities in corneas with subepithelial

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

    International Nuclear Information System (INIS)

    Liu, Yang; Ren, Li; Wang, Yingjun

    2014-01-01

    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

  1. 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.

  2. 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.

  3. The Impact of Type 1 Diabetes Mellitus on Corneal Epithelial Nerve Morphology and the Corneal Epithelium

    OpenAIRE

    Cai, Daniel; Zhu, Meifang; Petroll, W. Matthew; Koppaka, Vindhya; Robertson, Danielle M.

    2014-01-01

    Diabetic corneal neuropathy can result in chronic, sight-threatening corneal pathology. Although the exact etiology is unknown, it is believed that a reduction in corneal sensitivity and loss of neurotrophic support contributes to corneal disease. Information regarding the relationship between nerve loss and effects on the corneal epithelium is limited. We investigated changes in the corneal epithelium and nerve morphology using three-dimensional imaging in vivo and in situ in a streptozotoci...

  4. Construction of Anterior Hemi-Corneal Equivalents Using Nontransfected Human Corneal Cells and Transplantation in Dog Models.

    Science.gov (United States)

    Xu, Bin; Song, Zhan; Fan, Tingjun

    2017-11-01

    Tissue-engineered human anterior hemi-cornea (TE-aHC) is a promising equivalent for treating anterior lamellar keratopathy to surmount the severe shortage of donated corneas. This study was intended to construct a functional TE-aHC with nontransfected human corneal stromal (ntHCS) and epithelial (ntHCEP) cells using acellular porcine corneal stromata (aPCS) as a carrier scaffold, and evaluate its biological functions in a dog model. To construct a TE-aHC, ntHCS cells were injected into an aPCS scaffold and cultured for 3 days; then, ntHCEP cells were inoculated onto the Bowman's membrane of the scaffold and cultured for 5 days under air-liquid interface condition. After its morphology and histological structure were characterized, the constructed TE-aHC was transplanted into dog eyes via lamellar keratoplasty. The corneal transparency, thickness, intraocular pressure, epithelial integrity, and corneal regeneration were monitored in vivo, and the histological structure and histochemical property were examined ex vivo 360 days after surgery, respectively. The results showed that the constructed TE-aHC was highly transparent and composed of a corneal epithelium of 7-8 layer ntHCEP cells and a corneal stroma of regularly aligned collagen fibers and well-preserved glycosaminoglycans with sparsely distributed ntHCS cells, mimicking a normal anterior hemi-cornea (aHC). Moreover, both ntHCEP and ntHCS cells maintained positive expression of their marker and functional proteins. After transplantation into dog eyes, the constructed TE-aHC acted naturally in terms of morphology, structure and inherent property, and functioned well in maintaining corneal clarity, thickness, normal histological structure, and composition in dog models by reconstructing a normal aHC, which could be used as a promising aHC equivalent in corneal regenerative medicine and aHC disorder therapy. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

  5. Improvement In Rabbit Corneal Cell Suspension Viability After Freezing With Gingko Biloba Extrakt

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    Murad Aktan

    2007-01-01

    Full Text Available We investigated whether the addition of Gingko Biloba extract (EGb 761 to rabbit corneal epithelial medium before cell freezing improved cell viability after freezing then thawing. After removal of corneas, they were treated with enzymes and the corneal epithelium was prepared as a single cell suspension in freezing media with or without EGb 761. After freezing for two weeks then thawing, a higher cell viability was found in the cornea cell suspensions which had been frozen pretreated with EGb 761 in the media. The improvement with corneal cell viability with EGb 761 pretreatment is postulated to be based on the antioxidant capacity of the plant extract.

  6. 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.

  7. 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.

  8. A minimal model of epithelial tissue dynamics and its application to the corneal epithelium

    Science.gov (United States)

    Henkes, Silke; Matoz-Fernandez, Daniel; Kostanjevec, Kaja; Coburn, Luke; Sknepnek, Rastko; Collinson, J. Martin; Martens, Kirsten

    Epithelial cell sheets are characterized by a complex interplay of active drivers, including cell motility, cell division and extrusion. Here we construct a particle-based minimal model tissue with only division/death dynamics and show that it always corresponds to a liquid state with a single dynamic time scale set by the division rate, and that no glassy phase is possible. Building on this, we construct an in-silico model of the mammalian corneal epithelium as such a tissue confined to a hemisphere bordered by the limbal stem cell zone. With added cell motility dynamics we are able to explain the steady-state spiral migration on the cornea, including the central vortex defect, and quantitatively compare it to eyes obtained from mice that are X-inactivation mosaic for LacZ.

  9. Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

    Science.gov (United States)

    Turner, Helen C; Budak, Murat T; Akinci, M A Murat; Wolosin, J Mario

    2007-05-01

    To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that

  10. 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.

  11. In vitro expansion and characterization of corneal stem cells isolated from an eye with malignant melanoma.

    Science.gov (United States)

    Samoilă, O; Soriţău, Olga; Călugăru, M; Totu, Lăcrămioara; Şuşman, S; Cristian, Cristina; Mihu, Carmen Mihaela

    2013-01-01

    The objective of this study was the identification, characterization and in vitro replication of the human corneal stem cells, taking into consideration the difficulties in obtaining sufficient corneal material from living donors. The study explored a variety of stem cell markers, usually found in embryonic or adult mesenchymal stem cells. Culture medium and replication substrates had to be identified, with no data available on this subject in our country (there are no other reports on corneal stem cells in Romania, to our knowledge). Corneal epithelial limbus was harvested from an enucleated eye, containing also a choroid malignant melanoma. Stem cells from the limbus were isolated and cultivated in vitro. Expression of specific stem cell markers was evaluated with immunocytochemistry. Corneal stem cell expansion in primary culture was slow, achieving 70-80% confluence after 28 days. Stem cells were easily isolated in standard medium, showed fibroblastoid morphology and were positive for certain stem cell specific markers in immunocytochemical staining: Oct3÷4, SOX2, Nanog, SSEA4, CD44, CD90, CD133, and CD34. They also expressed pan-cytokeratin. Donor age (72 years) and the presence of a malignant tumor close to limbal stem niche could have had an impact on the proliferation rate and the characteristics of the corneal stem cells. Isolated limbal cells were adult type stem cells with an epithelial orientation. The characterization of these cells with immunocytochemistry allowed us to observe surface markers that other stem cells also express.

  12. Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane.

    Science.gov (United States)

    Rohaina, Che Man; Then, Kong Yong; Ng, Angela Min Hwei; Wan Abdul Halim, Wan Haslina; Zahidin, Aida Zairani Mohd; Saim, Aminuddin; Idrus, Ruszymah B H

    2014-03-01

    The cornea can be damaged by a variety of clinical disorders or chemical, mechanical, and thermal injuries. The objectives of this study were to induce bone marrow mesenchymal stem cells (BMSCs) to corneal lineage, to form a tissue engineered corneal substitute (TEC) using BMSCs, and to treat corneal surface defects in a limbal stem cell deficiency model. BMSCs were induced to corneal lineage using limbal medium for 10 days. Induced BMSCs demonstrated upregulation of corneal stem cell markers; β1-integrin, C/EBPδ, ABCG2, and p63, increased protein expression of CK3 and p63 significantly compared with the uninduced ones. For TEC formation, passage 1 BMSCs were trypsinized and seeded on amniotic membrane in a transwell co-culture system and were grown in limbal medium. Limbal stem cell deficiency models were induced by alkaline injury, and the TEC was implanted for 8 weeks. Serial slit lamp evaluation revealed remarkable improvement in corneal regeneration in terms of corneal clarity and reduced vascularization. Histologic and optical coherence tomography analyses demonstrated comparable corneal thickness and achieved stratified epithelium with a compact stromal layer resembling that of normal cornea. CK3 and p63 were expressed in the newly regenerated cornea. In conclusion, BMSCs can be induced into corneal epithelial lineage, and these cells are viable for the formation of TEC, to be used for the reconstruction of the corneal surface in the limbal stem cell deficient model. Copyright © 2014 Mosby, Inc. All rights reserved.

  13. Heterogeneity of limbal basal epithelial progenitor cells.

    Science.gov (United States)

    Hayashida, Yasutaka; Li, Wei; Chen, Ying-Ting; He, Hua; Chen, Szu-yu; Kheirkah, Ahmad; Zhu, Ying-Tien; Matsumoto, Yukihiro; Tseng, Scheffer C G

    2010-11-01

    Although corneal epithelial stem cells (SCs) are located at the limbus between the cornea and the conjunctiva, not all limbal basal epithelial cells are SCs. Using 2 dispase digestions to remove different amounts of limbal basal epithelial cells for cross-sections, flat mounts, and cytospin preparations, double immunostaining to pancytokeratins (PCK) and vimentin (Vim) identified 3 p63+ epithelial progenitors such as PCK-/Vim+, PCK/Vim, and PCK-/Vim+ and 1 p63+ mesenchymal cell, PCK-/Vim+. PCK-/Vim- progenitors had the smallest cell size were 10-20 times more enriched on collagen I-coated dishes in the 5-minute rapid adherent fraction that contained the highest percentage of p63+ cells but the lowest percentage of cytokeratin12+ cells, and gave rise to high Ki67 labeling and vivid clonal growth. In contrast, PCK+/Vim+ and PCK+/Vim- progenitors were found more in the slow-adherent fraction and yielded poor clonal growth. PCK/Vim progenitors and clusters of PCK-/Vim+ mesenchymal cells, which were neither melanocytes nor Langerhans cells, were located in the limbal basal region. Therefore, differential expression of PCK and Vim helps identify small PCK-/Vim- cells as the most likely candidate for SCs among a hierarchy of heterogeneous limbal basal progenitors, and their close association with PCK-/Vim+ presumed "niche" cells.

  14. 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

  15. The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns

    Science.gov (United States)

    Cejka, Cestmir; Holan, Vladimir; Trosan, Peter; Zajicova, Alena; Javorkova, Eliska; Cejkova, Jitka

    2016-01-01

    The aim of this study was to examine whether mesenchymal stem cells (MSCs) and/or corneal limbal epithelial stem cells (LSCs) influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH) were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs), with adipose tissue MSCs (Ad-MSCs), or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS), α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and vascular endothelial factor (VEGF) were low. The central corneal thickness (taken as an index of corneal hydration) increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-β1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties. PMID:27057279

  16. 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

  17. Silk Film Topography Directs Collective Epithelial Cell Migration

    Science.gov (United States)

    Rosenblatt, Mark I.

    2012-01-01

    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. PMID:23185573

  18. Polysaccharide Hydrogel Combined with Mesenchymal Stem Cells Promotes the Healing of Corneal Alkali Burn in Rats

    Science.gov (United States)

    Liu, Xun; Yu, Min; Yang, Chunbo; Li, Xiaorong

    2015-01-01

    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. PMID:25789487

  19. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  20. 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.

  1. Changes in corneal sensation, epithelial damage, and tear function after descemet stripping automated endothelial keratoplasty.

    Science.gov (United States)

    Hirayama, Yumiko; Satake, Yoshiyuki; Hirayama, Masatoshi; Shimazaki-Den, Seika; Konomi, Kenji; Shimazaki, Jun

    2013-09-01

    To study the ocular surface changes in eyes after Descemet stripping automated endothelial keratoplasty (DSAEK) compared with those after penetrating keratoplasty (PKP). This prospective study compared the changes in 31 eyes of 28 patients who underwent DSAEK (DSAEK group) with those in 15 disease-matched eyes of 15 patients who underwent PKP (PKP group). Corneal epithelial integrity was evaluated using a fluorescein staining score. Corneal sensation was measured with a Cochet-Bonnet esthesiometer. Tear function was evaluated using the Schirmer test, tear clearance test, tear function index, and tear break-up time. The postoperative fluorescein staining score was significantly higher in the PKP group than in the DSAEK group (P = 0.02). Postoperative corneal sensation was significantly better in the DSAEK group than in the PKP group (P sensation after DSAEK was significantly better than the preoperative value (P = 0.02). There were no statistically significant changes in the Schirmer test, tear clearance test, tear function index, or break-up time before and after the surgery in both the DSAEK and PKP groups. No significant differences were observed between the DSAEK and PKP groups after the surgery. Corneal sensation was preserved, and epithelial damage was less severe after DSAEK compared with PKP. Preservation of corneal sensation may contribute to the early recovery of visual function and long-term maintenance of ocular surface health after DSAEK.

  2. In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma

    Directory of Open Access Journals (Sweden)

    Xiao-Yong Liu

    2013-08-01

    Full Text Available AIM:To reconstruct the lamellar cornea using human amniotic epithelial (HAE cells and rabbit cornea stroma in vitro using tissue engineering technology.METHODS: Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC morphology was observed by Hematoxylin-eosin (HE staining; its ultrastructure was observed by transmission electron microscopy (TEM and scanning electron microscopy (SEM; corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy.RESULTS:HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay.CONCLUSION: Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.

  3. In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma.

    Science.gov (United States)

    Liu, Xiao-Yong; Chen, Jian; Zhou, Qing; Wu, Jing; Zhang, Xiao-Ling; Wang, Li; Qin, Xiao-Yan

    2013-01-01

    To reconstruct the lamellar cornea using human amniotic epithelial (HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology. Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC) morphology was observed by Hematoxylin-eosin (HE) staining; its ultrastructure was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy. HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay. Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.

  4. Metaherpetic corneal disease in a dog associated with partial limbal stem cell deficiency and neurotrophic keratitis.

    Science.gov (United States)

    Ledbetter, Eric C; Marfurt, Carl F; Dubielzig, Richard R

    2013-07-01

    To describe clinical, in vivo confocal microscopic, histopathologic, and immunohistochemical features of a dog with metaherpetic corneal disease that developed subsequent to a protracted episode of canine herpesvirus-1 (CHV-1) dendritic ulcerative keratitis. A 7-year-old, spayed-female, Miniature Schnauzer was treated for bilateral CHV-1 dendritic ulcerative keratitis. Following resolution of ulcerative keratitis, sectoral peripheral superficial corneal gray opacification, vascularization, and pigmentation slowly migrated centripetally to the axial cornea of both eyes. Corneal sensitivity measured with a Cochet-Bonnet esthesiometer was dramatically and persistently reduced. In vivo corneal confocal microscopic examination revealed regions of epithelium with a conjunctival phenotype. In these areas, the surface epithelium was thin, disorganized, and composed of hyper-reflective epithelial cells. Goblet cells and Langerhans cells were frequent, and the subbasal nerve plexus was completely absent or markedly diminished. Histopathologic abnormalities in the globes were restricted to the superficial cornea and included sectoral corneal conjunctivalization, increased anterior stromal spindle cells, and vascularization. Immunohistochemical evaluation of the corneas with anti-neurotublin antibody demonstrated attenuation of the epithelial and subbasal nerve plexuses with marked stromal hyperinnervation and increased numbers of morphologically abnormal neurites. Similar to herpes simplex virus keratitis in humans, CHV-1 ulcerative keratitis may be associated with the development of chronic degenerative corneal disease in dogs. In the described dog, this chronic corneal disease included progressive corneal opacification because of partial limbal stem cell deficiency and neurotrophic keratitis. Long-term monitoring of dogs following resolution of active CHV-1 keratitis may be indicated, particularly when ulcerations persist for an extended period. © 2012 American College of

  5. Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.

    Science.gov (United States)

    Diao, Jin-Mei; Pang, Xin; Qiu, Yue; Miao, Ying; Yu, Miao-Miao; Fan, Ting-Jun

    2015-03-01

    A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 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.

  7. Epithelial cell polarity, stem cells and cancer

    DEFF Research Database (Denmark)

    Martin-Belmonte, Fernando; Perez-Moreno, Mirna

    2011-01-01

    After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related......, deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis....

  8. 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. © 2014. Published by The Company of Biologists Ltd.

  9. SD-OCT analysis of regional epithelial thickness profiles in keratoconus, postoperative corneal ectasia, and normal eyes.

    Science.gov (United States)

    Rocha, Karolinne Maia; Perez-Straziota, Claudia E; Perez-Straziota, E; Stulting, R Doyle; Randleman, J Bradley

    2013-03-01

    To assess corneal microarchitecture and regional epithelial thickness profile in eyes with keratoconus, postoperative corneal ectasia (ectasia), and normal unoperated eyes (controls) using spectral-domain optical coherence tomography (SD-OCT). Regional corneal epithelial thickness profiles were measured with anterior segment SD-OCT (Optovue RTVue-100, Optovue Inc., Fremont, CA). Epithelial thickness was assessed at 21 points, 0.5 mm apart, across the central 6-mm of the corneal apex in the horizontal and vertical meridians. One hundred twenty eyes were evaluated, including 49 eyes from 29 patients with keratoconus, 32 eyes from 16 patients with ectasia, and 39 eyes from 21 control patients. Average epithelial thickness at the corneal apex was 41.18 ± 6.47 μm (range: 30 to 51 μm) for keratoconus, 46.5 ± 6.72 μm for ectasia (range: 34 to 60 μm), and 50.45 ± 3.92 μm for controls (range: 42 to 55 μm). Apical epithelial thickness was significantly thinner in eyes with keratoconus (P ectasia (P = .0007) than in controls. Epithelial thickness ranges in all other areas varied widely for keratoconus (range: 21 to 101 μm) and ectasia (range: 30 to 82 μm) compared to controls (range: 43 to 64) (P = .0063). SD-OCT demonstrated significant central and regional epithelial thickness profile differences between keratoconus, ectasia, and control eyes, with significant variability and unpredictability in ectatic eyes. This regional irregularity may necessitate direct epithelial thickness measurement for treatments where underlying stromal variations may be clinically relevant, including corneal collagen cross-linking or topography-guided ablations. Copyright 2013, SLACK Incorporated.

  10. 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.

  11. Effect of topical vitamin E on ethanol-induced corneal epithelial apoptosis.

    Science.gov (United States)

    Bilgihan, Kamil; Konuk, Onur; Hondur, Ahmet; Akyürek, Nalan; Ozogul, Candan; Hasanreisoglu, Berati

    2005-01-01

    Ethanol is used to loosen the corneal epithelium before photoablation in laser subepithelial keratomileusis (LASEK). In this study, the apoptotic index of corneal epithelium after ethanol exposure and the effects of topical vitamin E were evaluated. The study was performed on 28 rabbit eyes in four groups. Group 1 comprised the controls. In group 2, 20% ethanol was applied topically for 20 seconds. In group 3, topical vitamin E was applied following 20% ethanol application. In group 4, only topical vitamin E was applied. Apoptosis was evaluated with TUNEL assay and transmission electron microscopy. Epithelial apoptosis was detected in all specimens in group 2. No apoptosis was detected in other groups except for one eye in group 1. The apoptotic index in group 2 was statistically higher than other groups (P < .001).

  12. Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

    Directory of Open Access Journals (Sweden)

    Morley Steven D

    2009-01-01

    Full Text Available Abstract Background The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs that produce transient amplifying cells (TACs which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age. Results The initial pattern of corneal epithelial patches in XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function but stabilised after 39 weeks. Conclusion Striping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound

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

    Science.gov (United States)

    Tanaka, Yohei; Nakayama, Jun

    2016-01-01

    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. 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. 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/cm(2) irradiation (Psolar energy reaching the Earth is in the NIR region, which cannot be adequately blocked by eyewear and thus can induce eye damage with intensive or long-term exposure, protection from both UV and NIR radiation may prevent changes in gene expression and in turn eye damage.

  14. Spectral-Domain OCT Analysis of Regional Epithelial Thickness Profiles in Keratoconus, Postoperative Corneal Ectasia, and Normal Eyes

    Science.gov (United States)

    Rocha, Karolinne Maia; Straziota, Claudia Perez; Stulting, R. Doyle; Randleman, J. Bradley

    2014-01-01

    PURPOSE To assess corneal microarchitecture and regional epithelial thickness profile in eyes with keratoconus, postoperative corneal ectasia, and normal unoperated eyes using spectral-domain optical coherence tomography (SD OCT). METHODS Regional corneal epithelial thickness profiles of eyes with keratoconus (KC) and postoperative corneal ectasia (Ectasia) were measured with anterior segment SC OCT (Optovue RTVue-100, Optovue Inc., Fremont, CA) and compared retrospectively to those of normal eyes (Control). Epithelial thickness was assessed at 21 points, 0.5 mm apart, across the central 6-mm of the corneal apex in the horizontal and vertical meridians. RESULTS One hundred twenty eyes were evaluated, including 49 eyes from 29 patients with KC, 32 eyes from 16 patients with Ectasia, and 39 eyes from 21 control patients. Average epithelial thickness at the corneal apex was 41.18±6.47μm (range 30 to 51 μm) in eyes with KC, 46.5±6.72μm in eyes with ectasia (range 34 to 60 μm), and 50.45±3.92 μm in normal eyes (range 42 to 55 μm). Apical epithelial thickness was significantly thinner in eyes with KC (p ectasia (p=.0007) than it was in controls. Epithelial thickness ranges in all other areas varied widely for KC (SD, range 21 to 101 μm) and ectasia (SD, range 30 to 82 μm) compared to controls (SD, range 43 to 64), p = .0063 CONCLUSION Central epithelial thickness was, on average, significantly thinner in ectatic corneas compared to controls; however, both central and regional epithelial thickness was highly irregular and variable in corneas with keratoconus and postoperative corneal ectasia. These thickness variations may alter preoperative topographic features and measurements in unpredictable ways, especially steepest K values. Regional epithelial thickness cannot be assumed to be uniform in ectatic corneas and therefore may require direct measurement when considering treatments for which underlying stromal thickness is particularly important, such as

  15. Corneal endothelial cell density and morphology in healthy Turkish eyes.

    Science.gov (United States)

    Arıcı, Ceyhun; Arslan, Osman Sevki; Dikkaya, Funda

    2014-01-01

    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 Filipino eyes and higher than that described in Indian, Thai, and Iranian eyes.

  16. Corneal Epithelial Wound Healing Promoted by Verbascoside-Based Liposomal Eyedrops

    Directory of Open Access Journals (Sweden)

    Luigi Ambrosone

    2014-01-01

    Full Text Available Different liposomal formulations were prepared to identify those capable of forming eyedrops for corneal diseases. Liposomes with neutral or slightly positive surface charge interact very well with the cornea. Then these formulations were loaded with verbascoside to heal a burn of corneal epithelium induced by alkali. The cornea surface affected involved in wound was monitored as a function of time. Experimental results were modeled by balance equation between the rate of healing, due to the flow of phenylpropanoid, and growth of the wound. The results indicate a latency time of only three hours and furthermore the corneal epithelium heals in 48 hours. Thus, the topical administration of verbascoside appears to reduce the action time of cells, as verified by histochemical and immunofluorescence assays.

  17. Corneal Epithelial Wound Healing Promoted by Verbascoside-Based Liposomal Eyedrops

    Science.gov (United States)

    Ambrosone, Luigi; Guerra, Germano; Cinelli, Mariapia; Filippelli, Mariaelena; Mosca, Monica; Vizzarri, Francesco; Giorgio, Dario; Costagliola, Ciro

    2014-01-01

    Different liposomal formulations were prepared to identify those capable of forming eyedrops for corneal diseases. Liposomes with neutral or slightly positive surface charge interact very well with the cornea. Then these formulations were loaded with verbascoside to heal a burn of corneal epithelium induced by alkali. The cornea surface affected involved in wound was monitored as a function of time. Experimental results were modeled by balance equation between the rate of healing, due to the flow of phenylpropanoid, and growth of the wound. The results indicate a latency time of only three hours and furthermore the corneal epithelium heals in 48 hours. Thus, the topical administration of verbascoside appears to reduce the action time of cells, as verified by histochemical and immunofluorescence assays. PMID:25165705

  18. 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.

  19. A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations.

    Science.gov (United States)

    Miyashita, Hideyuki; Shimmura, Shigeto; Higa, Kazunari; Yoshida, Satoru; Kawakita, Tetsuya; Shimazaki, Jun; Tsubota, Kazuo

    2008-07-01

    Corneal epithelial cell sheets co-cultivated with feeder cells are used to reconstruct the ocular surface in stem cell-depleted eyes. The present study was conducted to investigate the optimal method of using feeder cells in the interest of preserving progenitor cells in cultivated sheets. We compared the phenotype and secondary colony forming efficiency (CFE) of cell sheets that were engineered using 3T3 feeder cells as a separate layer or as a contact layer. We also devised a novel "duplex feeder" system that consists of two separate layers of feeder cells. After cells reached confluence, cells were cultured at the air-liquid interface to allow full stratification. Stratified sheets were then analyzed using immunohistochemistry and secondary colony formation. Contact feeder cultures and duplex feeder cultures yielded epithelial sheets with small, cuboid basal cells with strong expression of keratin (K)3, K12, and K 15. Furthermore, only duplex feeder layers reproduced the basal K 15, suprabasal K12 limbal phenotype where epithelial stem cells reside. A similar effect was observed when cornea stroma-derived progenitor cells were used as feeder cells. Duplex feeder sheets also produced significantly more secondary colonies than cells dissociated from single layer sheets, suggesting that the duplex feeder system produces transplantable sheets with a higher yield of progenitor cells.

  20. Results of accelerated trans-epithelial corneal collagen cross-linking protocol for the treatment of progressive keratoconus

    Directory of Open Access Journals (Sweden)

    Chen-Xing Zhang

    2017-10-01

    Full Text Available AIM: To report the long-term clinical outcomes of accelerated trans-epithelial corneal cross-linking(CXLprotocols using KXL System(Avedro, USAin the treatment of progressive keratoconus.METHODS: Totally 52 patients(102 eyeswith progressive keratoconus between December 2014 and February 2017 \\〖maximum keratometry values(Kmax≤60.0D, minimum corneal thickness(Thk≥400m\\〗 were treated with an accelerate trans-epithelial CXL protocol(UV-A irradiation intensity 45mW/cm2 with a total fluence of 7.2J/cm2using KXL system(Avedro, USAin Southwest Hospital. The average follow-up time was 11.65mo(range: 9-26mo. Uncorrected distance visual acuity(UDVA, corrected distance visual acuity(CDVA, intra-ocular pressure(IOP, slit-lamp microscope examination, Kmax and average keratometry values(AveK, corneal stromal demarcation line depth and endothelial cell density(ECDwere evaluated.RESULTS: The 52 patients(102 eyeswere included in this research, male 36(70 eyesand female 16(32 eyes, average age was 19.5±4.6 years. Preoperative CDVA was 0.84±0.89(LogMAR, postoperative CDVA was 0.69±0.72(P=0.398. Preoperative UDVA was 1.02±0.62(LogMAR, postoperative UDVA was 0.85±0.59(P=0.154. Preoperative IOP was 12.95±4.40mmHg, postoperative IOP was 11.92±3.66mmHg(P=0.272. No statistical difference(P=0.552has been found between preoperative and postoperative ECD. Nevertheless, on the Sirius anterior system(Sirius, CSO, Itlay, significant statistical difference(P=0.017was confirmed between preoperative Kmax(50.83±3.48Dand postoperative Kmax(52.05±3.63D. Meanwhile, the postoperative Avek(47.74±2.51Dwas significantly lower(P=0.041than the preoperative Avek(48.73±4.33D. The average corneal stromal demarcation line depth(192±23.6μmwas detected by the anterior segment OCT. No statistical difference(P=0.816has been found between preoperative and postoperative Thk. No severe complication was observed in all cases.CONCLUSION: Accelerated trans-epithelial CXL was

  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. Expansion and cryopreservation of porcine and human corneal endothelial cells.

    Science.gov (United States)

    Marquez-Curtis, Leah A; McGann, Locksley E; Elliott, Janet A W

    2017-08-01

    Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me 2 SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me 2 SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  3. 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

  4. The role of endogenous epidermal growth factor receptor ligands in mediating corneal epithelial homeostasis.

    Science.gov (United States)

    Peterson, Joanne L; Phelps, Eric D; Doll, Mark A; Schaal, Shlomit; Ceresa, Brian P

    2014-05-01

    To provide a comprehensive study of the biological role and therapeutic potential of six endogenous epidermal growth factor receptor (EGFR) ligands in corneal epithelial homeostasis. Kinetic analysis and dose response curves were performed by using in vitro and in vivo wound-healing assays. Biochemical assays were used to determine receptor expression and activity. Human tears were collected and quantitatively analyzed by multianalyte profiling for endogenous EGFR ligands. Epidermal growth factor receptor ligands improved wound closure and activated EGFR, but betacellulin (BTC) was the most efficacious promoter of wound healing in vitro. In contrast, only epidermal growth factor (EGF) promoted wound healing in vivo. Human tears from 25 healthy individuals showed EGFR ligands at these average concentrations: EGF at 2053 ± 312.4 pg/mL, BTC at 207 ± 39.4 pg/mL, heparin-binding EGF at 44 ± 5.8 pg/mL, amphiregulin at 509 ± 28.8 pg/mL, transforming growth factor-α at 84 ± 19 pg/mL, and epiregulin at 52 ± 15 pg/mL. Under unwounded conditions, only EGF was present at concentrations near the ligand's Kd for the receptor, indicating it is the primary mediator of corneal epithelial homeostasis. Other ligands were present but at concentrations 11- to 7500-fold less their Kd, preventing significant ligand binding. Further, the high levels of EGF and its predicted binding preclude receptor occupancy by exogenous ligand and can explain the discrepancy between the in vitro and in vivo data. Therefore, therapeutic use of EGFR ligands may be unpredictable and impractical.

  5. Maintenance of the corneal epithelium is carried out by germinative cells of its basal stratum and not by presumed stem cells of the limbus

    Directory of Open Access Journals (Sweden)

    A. Haddad

    2014-06-01

    Full Text Available The purpose of this investigation was to analyze the proliferative behavior of rabbit corneal epithelium and establish if any particular region was preferentially involved in epithelial maintenance. [3H]-thymidine was injected intravitreally into both normal eyes and eyes with partially scraped corneal epithelium. Semithin sections of the anterior segment were evaluated by quantitative autoradiography. Segments with active replication (on and those with no cell division (off were intermingled in all regions of the tissue, suggesting that the renewal of the epithelial surface of the cornea followed an on/off alternating pattern. In the limbus, heavy labeling of the outermost layers was observed, coupled with a few or no labeled nuclei in the basal stratum. This suggests that this region is a site of rapid cell differentiation and does not contain many slow-cycling cells. The conspicuous and protracted labeling of the basal layer of the corneal epithelium suggests that its cells undergo repeated cycles of replication before being sent to the suprabasal strata. This replication model is prone to generate label-retaining cells. Thus, if these are adult stem cells, one must conclude that they reside in the corneal basal layer and not the limbal basal layer. One may also infer that the basal cells of the cornea and not of the limbus are the ones with the main burden of renewing the corneal epithelium. No particular role in this process could be assigned to the cells of the basal layer of the limbal epithelium.

  6. Development of Cell Analysis Software for Cultivated Corneal Endothelial Cells.

    Science.gov (United States)

    Okumura, Naoki; Ishida, Naoya; Kakutani, Kazuya; Hongo, Akane; Hiwa, Satoru; Hiroyasu, Tomoyuki; Koizumi, Noriko

    2017-11-01

    To develop analysis software for cultured human corneal endothelial cells (HCECs). Software was designed to recognize cell borders and to provide parameters such as cell density, coefficient of variation, and polygonality of cultured HCECs based on phase contrast images. Cultured HCECs with high or low cell density were incubated with Ca-free and Mg-free phosphate-buffered saline for 10 minutes to reveal the cell borders and were then analyzed with software (n = 50). Phase contrast images showed that cell borders were not distinctly outlined, but these borders became more distinctly outlined after phosphate-buffered saline treatment and were recognized by cell analysis software. The cell density value provided by software was similar to that obtained using manual cell counting by an experienced researcher. Morphometric parameters, such as the coefficient of variation and polygonality, were also produced by software, and these values were significantly correlated with cell density (Pearson correlation coefficients -0.62 and 0.63, respectively). The software described here provides morphometric information from phase contrast images, and it enables subjective and noninvasive quality assessment for tissue engineering therapy of the corneal endothelium.

  7. Restoration of Corneal Transparency by Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Sharad K. Mittal

    2016-10-01

    Full Text Available Transparency of the cornea is indispensable for optimal vision. Ocular trauma is a leading cause of corneal opacity, leading to 25 million cases of blindness annually. Recently, mesenchymal stem cells (MSCs have gained prominence due to their inflammation-suppressing and tissue repair functions. Here, we investigate the potential of MSCs to restore corneal transparency following ocular injury. Using an in vivo mouse model of ocular injury, we report that MSCs have the capacity to restore corneal transparency by secreting high levels of hepatocyte growth factor (HGF. Interestingly, our data also show that HGF alone can restore corneal transparency, an observation that has translational implications for the development of HGF-based therapy.

  8. Functional limbal epithelial cells can be successfully isolated from organ culture rims following long-term storage.

    Science.gov (United States)

    Tovell, Victoria E; Massie, Isobel; Kureshi, Alvena K; Daniels, Julie T

    2015-06-01

    Because of a shortage of fresh corneal tissue for research, it was of interest to investigate the potential of successfully isolating human limbal epithelial cells (hLECs) from organ culture corneal-scleral (OCCS) rims. Superficial segments of corneal limbus were dissected and digested using collagenase (0.5 mg/mL, 16 hours at 37 °C). Cell suspensions were separated into four different growth conditions: corneal epithelial cell medium (CM); CM + 3T3-Swiss albino cells; stromal stem cell medium (SM); and SM + 3T3 cells. Colony number, hLEC count, cell density, and colony forming efficiency (CFE) were quantified to assess different growth conditions. The expression profile associated with basal hLECs was assessed by immunofluorescence, and epithelial integrity was measured using our real architecture for 3D tissue (RAFT) corneal tissue equivalent. Human limbal epithelial cells can be successfully isolated from OCCS rims following 4 weeks in storage with an 80.55% success rate with 36 corneal rims. Stromal stem cell medium + 3T3s provided optimal growth conditions. Colony number, total cell number, and cell density were significantly higher at day 7 in cultures with SM than in CM. There were no significant differences between SM and CM when assessing CFE and the expression profile associated with basal hLECs. Cells maintained in SM were found to produce a higher quality epithelium than that cultured in CM. Organ culture corneal-scleral rims can be a valuable source for hLEC. Using a combination of collagenase-based isolation and medium designed for stromal stem cell isolation, a high number of good quality hLECs can be cultured from tissue that would have otherwise been ignored.

  9. Validation of Na,K-ATPase pump function of corneal endothelial cells for corneal regenerative medicine.

    Science.gov (United States)

    Hatou, Shin; Higa, Kazunari; Inagaki, Emi; Yoshida, Satoru; Kimura, Erika; Hayashi, Ryuhei; Tsujikawa, Motokazu; Tsubota, Kazuo; Nishida, Kohji; Shimmura, Shigeto

    2013-12-01

    Tissue-engineering approaches to cultivate corneal endothelial cells (CECs) or induce CECs from stem cells are under investigation for the treatment of endothelial dysfunction. Before clinical application, a validation method to determine the quality of these cells is required. In this study, we quantified the endothelial pump function required for maintaining the corneal thickness using rabbit CECs (RCECs) and a human CEC line (B4G12). The potential difference of RCECs cultured on a permeable polyester membrane (Snapwell), B4G12 cells on Snapwell, or B4G12 cells on a collagen membrane (CM6) was measured by an Ussing chamber system, and the effect of different concentrations of ouabain (Na,K-ATPase specific inhibitor) was obtained. A mathematical equation derived from the concentration curve revealed that 2 mM ouabain decreases pump function of RCECs to 1.0 mV, and 0.6 mM ouabain decreases pump function of B4G12 on CM6 to 1.0 mV. Ouabain injection into the anterior chamber of rabbit eyes at a concentration of pump function >1.0 mV is required to maintain the corneal thickness. These results can be used for standardization of CEC pump function and validation of tissue-engineered CEC sheets for clinical use.

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

    Science.gov (United States)

    Ozmen, Mehmet Cuneyt; Hondur, Ahmet; Yilmaz, Guldal; Bilgihan, Kamil; Hasanreisoglu, Berati

    2014-01-01

    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. 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. 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. 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.

  11. Histopathological features of equine superficial, nonhealing, corneal ulcers.

    Science.gov (United States)

    Hempstead, Julie E; Clode, Alison B; Borst, Luke B; Gilger, Brian C

    2014-07-01

    To evaluate corneal changes associated with chronic, nonhealing, superficial, corneal ulcers in horses via common histopathological stains. Retrospective study. Twenty-four horses diagnosed with chronic, nonhealing, superficial, corneal ulceration. The medical records of horses evaluated at North Carolina State University's Veterinary Teaching Hospital (NCSU-VTH) from 2005 to 2011, diagnosed with a chronic, nonhealing, superficial, corneal ulcer and treated with superficial keratectomy (SK) were reviewed. Inclusion criteria were superficial corneal ulceration, no cellular infiltration via slit-lamp biomicroscopy, no microorganisms evident on corneal cytology, and acquisition of samples for aerobic bacterial and common fungal cultures. Corneal tissue samples were evaluated histopathologically for the presence or absence of a nonadherent epithelial 'lip', epithelial dysmaturity, intraepithelial inflammatory cells, an acellular hyaline zone in the anterior stroma, and stromal inflammatory cells, fibrosis and vascularization. In the majority of analyzed samples, epithelial cells adjacent to the ulcerated site showed nonadherence to the basement membrane and dysmaturity. Intraepithelial inflammatory cell infiltration was uncommon. Histopathological features of an anterior stromal hyaline zone, intrastromal inflammation, fibrosis and vascularization were variably present. The most consistent histopathological characteristics of equine chronic, nonhealing, superficial, corneal ulcers include epithelial nonadherence, epithelial dysmaturity and mild to moderate stromal inflammation; however, one set of histopathological characteristics does not definitively define this syndrome in horses. Additionally, the anterior stromal acellular hyaline zone commonly cited in canine spontaneous chronic corneal epithelial defects (SCCED) is not a consistent finding in equine corneas. © 2013 American College of Veterinary Ophthalmologists.

  12. Karyotype changes in cultured human corneal endothelial cells

    OpenAIRE

    Miyai, Takashi; Maruyama, Yoko; Osakabe, Yasuhiro; Nejima, Ryohei; Miyata, Kazunori; Amano, Shiro

    2008-01-01

    Purpose To examine karyotype changes in cultured human corneal endothelial cells (HCECs). Methods HCECs with Descemet’s membrane were removed from 20 donors of various ages (range, 2–77 years; average, 43.7±26.4 years) and cultured on dishes coated with extracellular matrix produced by bovine corneal endothelial cells (BCECs). Karyotype changes were examined by G-band karyotyping of HCECs at the third passage from 12 donors and the fifth passage from 16 donors. The number of chromosomes was a...

  13. Trans-epithelial accelerated corneal cross-linking for keratoconus in children

    Science.gov (United States)

    Olivo-Payne, Andrew; Serna-Ojeda, Juan Carlos; Hernandez-Bogantes, Erick; Abdala-Figuerola, Alexandra; Pedro-Aguilar, Lucero; Lichtinger, Alejandro; Ramirez-Miranda, Arturo; Navas, Alejandro; Graue-Hernandez, Enrique O.

    2017-01-01

    The aim of the study is to evaluate the safety and efficacy of trans-epithelial accelerated corneal cross-linking (TE-ACXL) in children with progressive keratoconus. Retrospective, case-series of 23 eyes of 14 children who underwent TE-ACXL. Evaluations were performed at baseline and 1, 3, 6, 12 and 18mo postoperatively. Mean follow-up time of 23.82±3.15mo and mean age was 13.7±1.4y (range 11 to 16y). Mean preoperative uncorrected distance visual acuity changed from 0.92±0.45 logMAR (20/160) to 0.71±0.40 logMAR (20/100) (P=0.001). Mean keratometry (Km) changed from 53.87± 6.03 to 53.00±5.81 (P=0.001). Pachymetry did not have significant changes at last follow-up (P=0.30). The mean preoperative sphere was -5.58±2.48 and -4.89±4.66 D (P=0.11) at last follow-up; refractive cylinder from -5.58±2.48 to -5.02±2.23 (P=0.046). In conclusion, tomographic and refractive stability are shown in over 91% of eyes with pediatric progressive keratoconus who underwent TE-ACXL. PMID:29259913

  14. Trans-epithelial accelerated corneal cross-linking for keratoconus in children

    Directory of Open Access Journals (Sweden)

    Andrew Olivo-Payne

    2017-12-01

    Full Text Available The aim of the study is to evaluate the safety and efficacy of trans-epithelial accelerated corneal cross-linking (TE-ACXL in children with progressive keratoconus. Retrospective, case-series of 23 eyes of 14 children who underwent TE-ACXL. Evaluations were performed at baseline and 1, 3, 6, 12 and 18mo postoperatively. Mean follow-up time of 23.82±3.15mo and mean age was 13.7±1.4y (range 11 to 16y. Mean preoperative uncorrected distance visual acuity changed from 0.92±0.45 logMAR (20/160 to 0.71±0.40 logMAR (20/100 (P=0.001. Mean keratometry (Km changed from 53.87± 6.03 to 53.00±5.81 (P=0.001. Pachymetry did not have significant changes at last follow-up (P=0.30. The mean preoperative sphere was -5.58±2.48 and -4.89±4.66 D (P=0.11 at last follow-up; refractive cylinder from -5.58±2.48 to -5.02±2.23 (P=0.046. In conclusion, tomographic and refractive stability are shown in over 91% of eyes with pediatric progressive keratoconus who underwent TE-ACXL.

  15. NGF protects corneal, retinal, and cutaneous tissues/cells from phototoxic effect of UV exposure.

    Science.gov (United States)

    Rocco, Maria Luisa; Balzamino, Bijorn Omar; Aloe, Luigi; Micera, Alessandra

    2018-04-01

    Based on evidence that nerve growth factor (NGF) exerts healing action on damaged corneal, retinal, and cutaneous tissues, the present study sought to assess whether topical NGF application can prevent and/or protect epithelial cells from deleterious effects of ultraviolet (UV) radiation. Eyes from 40 young-adult Sprague Dawley rats and cutaneous tissues from 36 adult nude mice were exposed to UVA/B lamp for 60 min, either alone or in the presence of murine NGF. Corneal, retinal, and cutaneous tissues were sampled/processed for morphological, immunohistochemical, and biomolecular analysis, and results were compared statistically. UV exposure affected both biochemical and molecular expression of NGF and trkA NGFR in corneal, retinal, and cutaneous tissues while UV exposure coupled to NGF treatment enhanced NGF and trkA NGFR expression as well as reduced cell death. Overall, the findings of this in vivo/ex vivo study show the NGF ability to reduce the potential UV damage. Although the mechanism underneath this effect needs further investigation, these observations prospect the development of a pharmacological NGF-based therapy devoted to maintain cell function when exposed to phototoxic UV radiation.

  16. Phenotypic analyses of limbal epithelial cell cultures derived from donor corneoscleral rims.

    Science.gov (United States)

    Barnard, Z; Apel, A J; Harkin, D G

    2001-06-01

    Grafted cultures of limbal epithelial cells aid repair of the corneal epithelium, but their phenotype is unclear. In this study, the phenotype of cultures that were similar in age to those used clinically were analysed. Limbal epithelial cells were isolated from donor corneoscleral rims and grown in various media, including those designed for keratinocytes. Successful cultures in each medium developed predominantly small (10 microm) tightly packed cells. Immunocytochemistry and western blotting revealed expression of keratins 3, 14 and 19. Expression of these keratins in situ was confirmed by immunohistochemistry. Basal limbal epithelial cells were positive for keratins 14 and 19, and suprabasal cells were positive for keratin 3. However intense staining for keratin 14 was also observed at the inner cut edge of corneoscleral rims. These findings demonstrate the potential importance of keratins 14 and 19 as markers of epithelial cell differentiation in the human cornea.

  17. Therapeutic Effect of Human Adipose Tissue-Derived Mesenchymal Stem Cells in Experimental Corneal Failure Due to Limbal Stem Cell Niche Damage.

    Science.gov (United States)

    Galindo, Sara; Herreras, José M; López-Paniagua, Marina; Rey, Esther; de la Mata, Ana; Plata-Cordero, María; Calonge, Margarita; Nieto-Miguel, Teresa

    2017-10-01

    Limbal stem cells are responsible for the continuous renewal of the corneal epithelium. The destruction or dysfunction of these stem cells or their niche induces limbal stem cell deficiency (LSCD) leading to visual loss, chronic pain, and inflammation of the ocular surface. To restore the ocular surface in cases of bilateral LSCD, an extraocular source of stem cells is needed to avoid dependence on allogeneic limbal stem cells that are difficult to obtain, isolate, and culture. The aim of this work was to test the tolerance and the efficacy of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) to regenerate the ocular surface in two experimental models of LSCD that closely resemble different severity grades of the human pathology. hAT-MSCs transplanted to the ocular surface of the partial and total LSCD models developed in rabbits were well tolerated, migrated to inflamed tissues, reduced inflammation, and restrained the evolution of corneal neovascularization and corneal opacity. The expression profile of the corneal epithelial cell markers CK3 and E-cadherin, and the limbal epithelial cell markers CK15 and p63 was lost in the LSCD models, but was partially recovered after hAT-MSC transplantation. For the first time, we demonstrated that hAT-MSCs improve corneal and limbal epithelial phenotypes in animal LSCD models. These results support the potential use of hAT-MSCs as a novel treatment of ocular surface failure due to LSCD. hAT-MSCs represent an available, non-immunogenic source of stem cells that may provide therapeutic benefits in addition to reduce health care expenses. Stem Cells 2017;35:2160-2174. © 2017 AlphaMed Press.

  18. [Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells].

    Science.gov (United States)

    Kaji, Yuichi

    2005-11-01

    Corneal endothelial cell loss is a change that occurs with age, but its mechanism is still unclear. We postulated that interaction between advanced glycation end product(AGE) and its receptors is implicated in the corneal endothelial cell loss with age. We investigated the expression of AGE receptors: receptors for AGE(RAGE) and galectin-3 in bovine corneal endothelial cells by reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry. In addition, we investigated the effect of AGE on the cultured corneal endothelial cells. Expression of RAGE and galectin-3 was detected in bovine corneal endothelial cells. Galectin-3 was important in the internalization of AGE. In contrast, RAGE was important in the generation of reactive oxygen species and induction of apoptosis. Based on these data, the interaction of AGE in aqueous humor and AGE receptors expressed on the corneal endothelial cells was speculated to have a role in the corneal endothelial cell loss with age.

  19. 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.

  20. Transplantation with cultured stem cells derived from the human amniotic membrane for corneal alkali burns: an experimental study.

    Science.gov (United States)

    Zeng, Wei; Li, Yanwei; Zeng, Guangwei; Yang, Bo; Zhu, Yu

    2014-01-01

    Amniotic membranes (AM) have been used in a wide range of clinical applications. We successfully extracted mesenchymal stem cells (MSCs) from human AM, but little is known about the use and efficacy of human amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) for the treatment of alkali burns. We utilized hAM-dMSCs transplantation, AM grafting, and their combined use in the treatment of alkali burns. An experimental model in rabbits was devised to analyze the use of these techniques with immunocytochemistry and ELISA. The survival and migration of hAM-dMSCs labeled by SPION in the host were assessed with Prussian blue staining. Compared with the control group, the treated groups demonstrated faster reconstruction of the corneal epithelium, and lower levels of corneal opacification and neovascularization within corneal alkali burns. Furthermore, dark blue-stained particles were detected in the limbus corneae at day 28. These results demonstrated the ability of hAM-dMSCs to enhance epithelial healing and reduce corneal opacification and neovascularization in corneal alkali wounds.

  1. Radiation biology of human mammary epithelial cells

    International Nuclear Information System (INIS)

    Smith, H.S.; Yang, T.C.; Stampfer, M.R.; Hackett, A.J.

    1982-01-01

    Techniques have been developed for growing mass cultures of normal mammary epithelial cells (from reduction mammoplasties) and, most recently, for growing mammary epithelial cells in a highly efficient clonal assay. The availability of this clonal assay has enabled us to examine the dose-response curves for x rays

  2. Glycocalyx of lung epithelial cells.

    Science.gov (United States)

    Martins, Maria de Fátima; Bairos, Vasco A

    2002-01-01

    Due to their diversity and external location on cell membranes, glycans, as glycocalyx components, are key elements in eukaryotic cell, tissue, and organ homeostasis. Although information on the lung glycocalyx is scarce, this article aims to review, discuss, and summarize what is known about bronchoalveolar glycocalyx composition, mainly the sialic acids. It was deemed relevant, however, to make a brief introductory overview of the cell glycocalyx and its particular development in epithelial cells. After that, follows a summary of the evolution of the knowledge regarding the bronchoalveolar glycocalyx composition throughout the years, particularly its morphological features. Since sialic acids are located terminally on the bronchoalveolar lining cells' glycocalyx and play crucial roles, we focused mainly on the existing lung histochemical and biochemical data of these sugar residues, as well as their evolution throughout lung development. The functions of the lung glycocalyx sialic acids are discussed and interpretations of their roles analyzed, including those related to the negative overall superficial shield provided by these molecules. The increasing presence of these sugar residues throughout postnatal lung development should be regarded as pivotal in the development and maintenance of a dynamic bronchoalveolar architecture, supporting the normal histophysiology of the respiratory system. The case for a profound knowledge of lung glycocalyx--given its potential to provide answers to serious clinical problems--is made with particular reference to cystic fibrosis. Finally, concluding remarks and perspectives for future research in this field are put forth.

  3. Epithelial Cells in Urine: MedlinePlus Lab Test Information

    Science.gov (United States)

    ... page: https://medlineplus.gov/labtests/epithelialcellsinurine.html Epithelial Cells in Urine To use the sharing features on ... page, please enable JavaScript. What is an Epithelial Cells in Urine Test? Epithelial cells are a type ...

  4. 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.

  5. Corneal squamous cell carcinoma in a Border Collie.

    Science.gov (United States)

    Busse, Claudia; Sansom, Jane; Dubielzig, R R; Hayes, Alison

    2008-01-01

    A 6-year-old, female, spayed Border Collie was presented to the Unit of Comparative Ophthalmology at the Animal Health Trust with a 6-month history of a progressive nonpainful opacity of the left cornea. A keratectomy was performed and the tissue submitted for histopathology. The diagnosis was squamous cell carcinoma. There has been no recurrence of the neoplasm to date (5 months). Canine corneal squamous cell carcinoma (SCC) has not been reported previously in the UK.

  6. Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

    Directory of Open Access Journals (Sweden)

    Kathryn L McCabe

    Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

  7. 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; Forrest, Alistair R. R.; Rehli, Michael; Baillie, J. Kenneth; de Hoon, Michiel J. L.; Haberle, Vanja; Lassmann, Timo; Kulakovskiy, Ivan V.; Lizio, Marina; Andersson, Robin; Mungall, Christopher J.; Meehan, Terrence F.; Schmeier, Sebastian; Bertin, Nicolas; Jørgensen, Mette; Dimont, Emmanuel; Arner, Erik; Schmidl, Christian; Schaefer, Ulf; Medvedeva, Yulia A.; Plessy, Charles; Vitezic, Morana; Severin, Jessica; Semple, Colin A.; Ishizu, Yuri; Francescatto, Margherita; Alam, Intikhab; Albanese, Davide; Altschuler, Gabriel M.; Archer, John A. C.; Arner, Peter; Babina, Magda; Baker, Sarah; Balwierz, Piotr J.; Beckhouse, Anthony G.; Pradhan-Bhatt, Swati; Blake, Judith A.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Burroughs, A. Maxwell; Califano, Andrea; Cannistraci, Carlo V.; Carbajo, Daniel; Chen, Yun; Chierici, Marco; Ciani, Yari; Clevers, Hans C.; Dalla, Emiliano; Davis, Carrie A.; Detmar, Michael; Diehl, Alexander D.; Dohi, Taeko; Drabløs, Finn; Edge, Albert S. B.; Edinger, Matthias; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Fagiolini, Michela; Fairbairn, Lynsey; Fang, Hai; Farach-Carson, Mary C.; Faulkner, Geoffrey J.; Favorov, Alexander V.; Fisher, Malcolm E.; Frith, Martin C.; Fujita, Rie; Fukuda, Shiro; Furlanello, Cesare; Furuno, Masaaki; Furusawa, Jun-ichi; Geijtenbeek, Teunis B.; Gibson, Andrew; Gingeras, Thomas; Goldowitz, Daniel; Gough, Julian; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J.; Hamaguchi, Masahide; Hara, Mitsuko; Harbers, Matthias; Harshbarger, Jayson; Hasegawa, Akira; Hasegawa, Yuki; Hashimoto, Takehiro; Herlyn, Meenhard; Hitchens, Kelly J.; Ho Sui, Shannan J.; Hofmann, Oliver M.; Hoof, Ilka; Hori, Fumi; Huminiecki, Lukasz; Iida, Kei; Ikawa, Tomokatsu; Jankovic, Boris R.; Jia, Hui; Joshi, Anagha; Jurman, Giuseppe; Kaczkowski, Bogumil; Kai, Chieko; Kaida, Kaoru; Kaiho, Ai; Kajiyama, Kazuhiro; Kanamori-Katayama, Mutsumi; Kasianov, Artem S.; Kasukawa, Takeya; Katayama, Shintaro; Kato, Sachi; Kawaguchi, Shuji; Kawamoto, Hiroshi; Kawamura, Yuki I.; Kawashima, Tsugumi; Kempfle, Judith S.; Kenna, Tony J.; Kere, Juha; Khachigian, Levon M.; Kitamura, Toshio; Klinken, S. Peter; Knox, Alan J.; Kojima, Miki; Kojima, Soichi; Kondo, Naoto; Koseki, Haruhiko; Koyasu, Shigeo; Krampitz, Sarah; Kubosaki, Atsutaka; Kwon, Andrew T.; Laros, Jeroen F. J.; Lee, Weonju; Lennartsson, Andreas; Li, Kang; Lilje, Berit; Lipovich, Leonard; Mackay-sim, Alan; Manabe, Ri-ichiroh; Mar, Jessica C.; Marchand, Benoit; Mathelier, Anthony; Mejhert, Niklas; Meynert, Alison; Mizuno, Yosuke; Morais, David A. de Lima; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Motakis, Efthymios; Motohashi, Hozumi; Mummery, Christine L.; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nakazato, Kenichi; van Nimwegen, Erik; Ninomiya, Noriko; Nishiyori, Hiromi; Noma, Shohei; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A.; Pain, Arnab; Passier, Robert; Patrikakis, Margaret; Persson, Helena; Piazza, Silvano; Prendergast, James G. D.; Rackham, Owen J. L.; Ramilowski, Jordan A.; Rashid, Mamoon; Ravasi, Timothy; Rizzu, Patrizia; Roncador, Marco; Roy, Sugata; Rye, Morten B.; Saijyo, Eri; Sajantila, Antti; Saka, Akiko; Sakaguchi, Shimon; Sakai, Mizuho; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schneider, Claudio; Schultes, Erik A.; Schulze-Tanzil, Gundula G.; Schwegmann, Anita; Sengstag, Thierry; Sheng, Guojun; Shimoji, Hisashi; Shimoni, Yishai; Shin, Jay W.; Simon, Christophe; Sugiyama, Daisuke; Sugiyama, Takaaki; Suzuki, Masanori; Swoboda, Rolf K.; 't Hoen, Peter A. C.; Tagami, Michihira; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tatum, Zuotian; Thompson, Mark; Toyoda, Hiroo; Toyoda, Tetsuro; Valen, Eivind; van de Wetering, Marc; van den Berg, Linda M.; Verardo, Roberto; Vijayan, Dipti; Vorontsov, Ilya E.; Wasserman, Wyeth W.; Watanabe, Shoko; Wells, Christine A.; Winteringham, Louise N.; Wolvetang, Ernst; Wood, Emily J.; Yamaguchi, Yoko; Yamamoto, Masayuki; Yoneda, Misako; Yonekura, Yohei; Yoshida, Shigehiro; Zabierowski, Suzan E.; Zhang, Peter G.; Zhao, Xiaobei; Zucchelli, Silvia; Summers, Kim M.; Suzuki, Harukazu; Daub, Carsten O.; Kawai, Jun; Heutink, Peter; Hide, Winston; Freeman, Tom C.; Lenhard, Boris; Bajic, Vladimir B.; Taylor, Martin S.; Makeev, Vsevolod J.; Sandelin, Albin; Hume, David A.; Carninci, Piero

    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

  8. 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

  9. 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.

  10. Intracellular Position of Centrioles and the Direction of Homeostatic Epithelial Cell Movements in the Mouse Cornea.

    Science.gov (United States)

    Silverman, Erika; Zhao, Jin; Merriam, John C; Nagasaki, Takayuki

    2017-02-01

    Corneal epithelial cells exhibit continuous centripetal movements at a rate of about 30 µm per day, but neither the driving force nor the mechanism that determines the direction of movements is known. To facilitate the investigation of homeostatic cell movement, we examined if the intracellular position of a centriole can be used as a directional marker of epithelial cell movements in the mouse cornea. A direction of cell movements was estimated in fixed specimens from a pattern of underlying subepithelial nerve fibers. Intracellular position of centrioles was determined by gamma-tubulin immunohistology and plotted in a narrow strip along the entire diameter of a cornea from limbus to limbus. When we determined the position of centrioles in the peripheral cornea where cell movements proceed generally along a radial path, about 55% of basal epithelial cells contained a centriole in the front half of a cell. However, in the central cornea where cells exhibit a spiral pattern of movements, centrioles were distributed randomly. These results suggest that centrioles tend to be positioned toward the direction of movement in corneal basal epithelial cells when they are moving centripetally at a steady rate.

  11. Corneal Epithelial Thickness Measured by Manual Electronic Caliper Spectral Domain Optical Coherence Tomography: Distributions and Demographic Correlations in Preoperative Refractive Surgery Patients.

    Science.gov (United States)

    Rush, Sloan W; Matulich, Jennifer; Biskup, Joel; Cofoid, Philip; Rush, Ryan B

    2016-01-01

    The aim of this study was to report the distributions and demographic correlations of corneal epithelial thickness measured by manual electronic caliper spectral domain optical coherence tomography in preoperative refractive surgery patients. This was a retrospective review. The charts of 218 consecutive patients (413 eyes) who presented for refractive surgery evaluation from April 2013 through September 2013 were retrospectively reviewed. The mean corneal epithelial thickness was 51.0 μm with a range of 43 to 61 μm. Corneal epithelial thickness was significantly correlated with sex (P caliper optical coherence tomography in preoperative refractive surgery patients is comparable with the findings for the general population using other measurement techniques, the awareness of which may be useful in the preoperative assessment of these patients.

  12. The regenerative potential of epithelial stem cells in tissue repair.

    Science.gov (United States)

    Arandjelovic, Philip; Kaur, Pritinder

    2014-11-01

    Acute and chronic wounds encompass devastating injuries with significant physical, emotional and economic costs at both the individual and societal level. The pathogenesis of chronic wounds is as varied as the potential causes; however, contributing factors include repetitive ischaemia/reperfusion injury coupled with bacterial infection, inflammation and matrix degradation at the wound site. Similarly, the acute physical damage of burns may leave patients vulnerable to dehydration and infection, and in certain cases this may be followed by a body-wide systemic response with debilitating consequences. Epithelial stem cells provide a promising avenue for the treatment of burns and chronic wounds. This is exemplified by recent achievements such as the restoration of corneal epithelium using limbal stem cells, and the treatment of epidermolysis bullosa via a gene therapy approach. Nevertheless, many technical and regulatory challenges remain to be addressed. This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

    1982-01-01

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  14. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    Science.gov (United States)

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-08

    During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Effect of basic fibroblast growth factor and cytochrome c peroxidase combination in transgenic mice corneal epithelial healing process after excimer laser photoablation

    Directory of Open Access Journals (Sweden)

    Sergio Zaccaria Scalinci

    2011-02-01

    Full Text Available Sergio Zaccaria Scalinci1, Lucia Scorolli1, Alessandro Meduri2, Pier Luigi Grenga3, Giulia Corradetti1, Cristian Metrangolo11Low Vision Center – University of Bologna, Bologna, Italy; 2Department of Surgical Specialities, Ophthalmology Clinic, University of Messina, Messina, Italy; 3Department of Ophthalmology, University of Rome "La Sapienza", Rome, ItalyPurpose: To evaluate the role of prepared basic fibroblast growth factor (bFGF and cytochrome c peroxidase (CCP combination eyedrops in corneal epithelial healing of transgenic mice (B6(A-Rperd12/J after excimer laser photoablation. Materials and methods: In this prospective study, 216 eyes of 108 mice underwent bilateral photorefractive keratectomy. We considered 4 groups: A, B, C, and D. Group A received standard topical postoperative therapy with tobramycin, diclofenac, and dexamethasone eyedrops plus CCP at 3 drops per day for a week or until corneal re-epithelialization was achieved. Group B received standard topical postoperative therapy plus bFGF eyedrops and phosphate-buffered saline (PBS 3 drops per day for a week or until corneal re-epithelialization was complete. In group C, 1 eye received standard topical postoperative therapy plus CCP eyedrops, bFGF eyedrops, and PBS 3 drops per day for a week or until corneal re-epithelialization was complete. Control eyes (group D received a standard topical postoperative therapy plus placebo eyedrops. Mice were followed-up for a week from the day after the surgery to evaluate the rate of corneal re-epithelialization.Results: Data were analyzed by ANOVA using the XLSTAT 2010 software. Eyes in group A, B, and C healed completely before the fifth postoperative day, achieving, respectively, a re-epithelialization time of 92 hours ± 10 SD, 90 hours ± 12 SD, and 86 hours ± 12 SD. Group D had a re-epithelialization time of 121 hours ± 8 SD (P < 0.05. No side effects or toxic effects were documented.Conclusions: Results suggest that re-epithelialization

  16. 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.

  17. Density-gradient centrifugation enables the purification of cultured corneal endothelial cells for cell therapy by eliminating senescent cells

    Science.gov (United States)

    Okumura, Naoki; Kusakabe, Ayaka; Hirano, Hiroatsu; Inoue, Ryota; Okazaki, Yugo; Nakano, Shinichiro; Kinoshita, Shigeru; Koizumi, Noriko

    2015-01-01

    The corneal endothelium is essential for maintaining corneal transparency; therefore, corneal endothelial dysfunction causes serious vision loss. Tissue engineering-based therapy is potentially a less invasive and more effective therapeutic modality. We recently started a first-in-man clinical trial of cell-based therapy for treating corneal endothelial dysfunction in Japan. However, the senescence of corneal endothelial cells (CECs) during the serial passage culture needed to obtain massive quantities of cells for clinical use is a serious technical obstacle preventing the push of this regenerative therapy to clinical settings. Here, we show evidence from an animal model confirming that senescent cells are less effective in cell therapy. In addition, we propose that density-gradient centrifugation can eliminate the senescent cells and purify high potency CECs for clinical use. This simple technique might be applicable for other types of cells in the settings of regenerative medicine. PMID:26443440

  18. Research advances on tissue-engineered corneal endothelial cells transplantation

    Directory of Open Access Journals (Sweden)

    Si-Jie Zhao

    2015-02-01

    Full Text Available Due to the serious shortage of donor cornea materials and the donor limit, clinical popularization of penetrating keratoplasty is severely restricted. It is a hot spot of current research that applying tissue engineering in vitro to culture corneal endothelial cells(CECwith high density, regular hexagonal shape and healthy endothelial function. In this article, we reviewed the latest progress in the study of source of CEC seeder cells, selection of cultivating carries, type of CEC transplantation and immune mechanism that summarized the current research problems and made a prospect to the future.

  19. Effects of topical vitamin E on corneal superoxide dismutase, glutathione peroxidase activities and polymorphonuclear leucocyte infiltration after photorefractive keratectomy.

    Science.gov (United States)

    Bilgihan, Ayse; Bilgihan, Kamil; Yis, Ozgür; Sezer, Cem; Akyol, Gülen; Hasanreisoglu, Berati

    2003-04-01

    Photorefractive keratectomy (PRK) induces free radical formation and polymorphonuclear (PMN) cell infiltration in the cornea. Vitamin E is a free radical scavenger and protects the cells from reactive oxygen species. We investigated the effects of topical vitamin E on corneal PMN cell infiltration and corneal antioxidant enzyme activities after PRK. We studied four groups, each consisting of seven eyes. Group 1 were control eyes. In group 2 the corneal epithelium was removed by a blunt spatula (epithelial scrape). In group 3, corneal photoablation (59 micro m, 5 dioptres) was performed after epithelial removal (traditional PRK). In group 4 we tested the effects of topical Vitamin E after traditional PRK. Corneal tissues were removed and studied with enzymatic analysis (measurement of corneal superoxide dismutase and glutathione peroxidase activities) and histologically. Stromal PMN leucocyte counts were significantly higher after mechanical epithelial removal and traditional PRK (p < 0.05). Corneal superoxide dismutase and glutathione peroxidase activities decreased significantly after mechanical epithelial removal and traditional PRK (p < 0.05). In group 4, treated with vitamin E, corneal superoxide dismutase activity did not differ significantly from that in the medically non-treated groups, nor did corneal PMN cell infiltration after traditional PRK. The reduction of corneal glutathione peroxidase activity after PRK was reduced significantly after topical vitamin E treatment. Topical vitamin E treatment may be useful for reducing the harmful effects of reactive oxygen radical after epithelial scraping and PRK in that it increases corneal glutathione peroxidase activity.

  20. 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

  1. 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.

  2. 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

  3. 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 conclu....... 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....

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

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    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.

  5. 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

  6. 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.

  7. 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

  8. Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

    Science.gov (United States)

    Di Girolamo, N; Bobba, S; Raviraj, V; Delic, N C; Slapetova, I; Nicovich, P R; Halliday, G M; Wakefield, D; Whan, R; Lyons, J G

    2015-01-01

    Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation. © 2014 AlphaMed Press.

  9. 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. © 2013 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  10. 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....

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

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    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.

  12. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    Science.gov (United States)

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  13. Corneal regeneration by induced human buccal mucosa cultivated on an amniotic membrane following alkaline injury.

    Science.gov (United States)

    Man, Rohaina Che; Yong, Then Kong; Hwei, Ng Min; Halim, Wan Haslina Wan Abdul; Zahidin, Aida Zairani Mohd; Ramli, Roszalina; Saim, Aminuddin Bin; Idrus, Ruszymah Binti Hj

    2017-01-01

    Various clinical disorders and injuries, such as chemical, thermal, or mechanical injuries, may lead to corneal loss that results in blindness. PURPOSE : The aims of this study were to differentiate human buccal mucosa (BMuc) into corneal epithelial-like cells, to fabricate engineered corneal tissue using buccal mucosal epithelial cells, and to reconstruct a damaged corneal epithelium in a nude rat model. BMuc were subjected to 10 d of induction factors to investigate the potential of cells to differentiate into corneal lineages. Corneal stem cell markers β1-integrin, C/EBPδ, ABCG2, p63, and CK3 were upregulated in the gene expression analysis in induced BMuc, whereas CK3 and p63 showed significant protein expression in induced BMuc compared to the uninduced cells. BMuc were then left to reach 80% confluency after differential trypsinization. The cells were harvested and cultivated on a commercially available untreated air-dried amniotic membrane (AM) in a Transwell system in induction medium. The corneal constructs were fabricated and then implanted into damaged rat corneas for up to 8 weeks. A significant improvement was detected in the treatment group at 8 weeks post-implantation, as revealed by slit lamp biomicroscopy analysis. The structure and thickness of the corneal layer were also analyzed using histological staining and time-domain optical coherence tomography scans and were found to resemble a native corneal layer. The protein expression for CK3 and p63 were continuously detected throughout the corneal epithelial layer in the corneal construct. In conclusion, human BMuc can be induced to express a corneal epithelial-like phenotype. The addition of BMuc improves corneal clarity, prevents vascularization, increases corneal thickness and stromal alignment, and appears to have no adverse effect on the host after implantation.

  14. Postnatal periodontal ligament as a novel adult stem cell source for regenerative corneal cell therapy.

    Science.gov (United States)

    Yam, Gary Hin-Fai; Teo, Ericia Pei-Wen; Setiawan, Melina; Lovatt, Matthew J; Yusoff, Nur Zahirah Binte M; Fuest, Matthias; Goh, Bee-Tin; Mehta, Jodhbir S

    2018-03-13

    Corneal opacities are a leading cause of global blindness. They are conventionally treated by the transplantation of donor corneal tissue, which is, restricted by a worldwide donor material shortage and allograft rejection. Autologous adult stem cells with a potential to differentiate into corneal stromal keratocytes (CSKs) could offer a suitable choice of cells for regenerative cell therapy. Postnatal periodontal ligament (PDL) contains a population of adult stem cells, which has a similar embryological origin as CSK, that is cranial neural crest. We harvested PDL cells from young adult teeth extracted because of non-functional or orthodontic reason and differentiated them towards CSK phenotype using a two-step protocol with spheroid formation followed by growth factor and cytokine induction in a stromal environment (human amnion stroma and porcine corneal stroma). Our results showed that the PDL-differentiated CSK-like cells expressed CSK markers (CD34, ALDH3A1, keratocan, lumican, CHST6, B3GNT7 and Col8A2) and had minimal expression of genes related to fibrosis and other lineages (vasculogenesis, adipogenesis, myogenesis, epitheliogenesis, neurogenesis and hematogenesis). Introduction of PDL spheroids into the stroma of porcine corneas resulted in extensive migration of cells inside the host stroma after 14-day organ culture. Their quiescent nature and uniform cell distribution resembled to that of mature CSKs inside the native stroma. Our results demonstrated the potential translation of PDL cells for regenerative corneal cell therapy for corneal opacities. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  15. BVES regulates EMT in human corneal and colon cancer cells and is silenced via promoter methylation in human colorectal carcinoma.

    Science.gov (United States)

    Williams, Christopher S; Zhang, Baolin; Smith, J Joshua; Jayagopal, Ashwath; Barrett, Caitlyn W; Pino, Christopher; Russ, Patricia; Presley, Sai H; Peng, DunFa; Rosenblatt, Daniel O; Haselton, Frederick R; Yang, Jin-Long; Washington, M Kay; Chen, Xi; Eschrich, Steven; Yeatman, Timothy J; El-Rifai, Wael; Beauchamp, R Daniel; Chang, Min S

    2011-10-01

    The acquisition of a mesenchymal phenotype is a critical step in the metastatic progression of epithelial carcinomas. Adherens junctions (AJs) are required for suppressing this epithelial-mesenchymal transition (EMT) but less is known about the role of tight junctions (TJs) in this process. Here, we investigated the functions of blood vessel epicardial substance (BVES, also known as POPDC1 and POP1), an integral membrane protein that regulates TJ formation. BVES was found to be underexpressed in all stages of human colorectal carcinoma (CRC) and in adenomatous polyps, indicating its suppression occurs early in transformation. Similarly, the majority of CRC cell lines tested exhibited decreased BVES expression and promoter DNA hypermethylation, a modification associated with transcriptional silencing. Treatment with a DNA-demethylating agent restored BVES expression in CRC cell lines, indicating that methylation represses BVES expression. Reexpression of BVES in CRC cell lines promoted an epithelial phenotype, featuring decreased proliferation, migration, invasion, and anchorage-independent growth; impaired growth of an orthotopic xenograft; and blocked metastasis. Conversely, interfering with BVES function by expressing a dominant-negative mutant in human corneal epithelial cells induced mesenchymal features. These biological outcomes were associated with changes in AJ and TJ composition and related signaling. Therefore, BVES prevents EMT, and its epigenetic silencing may be an important step in promoting EMT programs during colon carcinogenesis.

  16. 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...

  17. Corneal Protection for Burn Patients

    Science.gov (United States)

    2013-10-01

    Create ectropion by blepharoplasty in New Zealand white rabbits. Assess epithelial defects and corneal ulcers . Measure inflammatory cytokines and...7, with corneal ulceration developing in the most severe cases by week 3. Histopathological results revealed epithelium infiltrated by heterophilic...excellent surgical model of evaporative dry eye. Damage to the cornea and conjunctiva manifesting as punctate epithelial erosions and corneal ulceration is

  18. ROCK inhibitor enhances adhesion and wound healing of human corneal endothelial cells.

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    Aurélien Pipparelli

    Full Text Available Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34, the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

  19. Correlation analysis of reject reaction and corneal endothelial cells after penetrating keratoplasty from donation after cardiac death

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    Qian Cao

    2017-01-01

    Full Text Available AIM: To study the relativity between reject reaction from donation after cardiac death(DCDand corneal endothelial cell source of corneal graft after penetrating keratoplasty.METHODS:Totally 28 cases of corneal graft rejection after penetrating keratoplasty with cardiac death donor cornea were analyzed using corneal endothelial microscope at less than 1mo, 2-3mo, 4-6mo, 7-12mo after operation.RESULTS:Coefficient variation of corneal endothelial cell of the 28 cases at less than 1mo,2-3mo, 4-6mo and 7-12mo were 38.23%, 49.56%, 57.18%, 65.04%. Corneal endothelial cell density were 2071.15±311.47, 1771.33±348.18, 1626.59±353.92, 1553.14±307.31. The coefficient variation of corneal endothelial cells was positively correlated with rejection(r=0.95, Pr=-0.93, PCONCLUSION: The corneal endothelial cell coefficient variation increased gradually and the corneal endothelial cell density decreased gradually after DCD corneal allograft rejection. Corneal endothelial cell coefficient variation and corneal endothelial cell density can be used as indicators of early detection of postoperative rejection.

  20. 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

  1. 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.

  2. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells

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    Park Tae

    2008-12-01

    Full Text Available Abstract Background Acinetobacter baumannii is a nosocomial pathogen of increasing importance, but the pathogenic mechanism of this microorganism has not been fully explored. This study investigated the potential of A. baumannii to invade epithelial cells and determined the role of A. baumannii outer membrane protein A (AbOmpA in interactions with epithelial cells. Results A. baumannii invaded epithelial cells by a zipper-like mechanism, which is associated with microfilament- and microtubule-dependent uptake mechanisms. Internalized bacteria were located in the membrane-bound vacuoles. Pretreatment of recombinant AbOmpA significantly inhibited the adherence to and invasion of A. baumannii in epithelial cells. Cell invasion of isogenic AbOmpA- mutant significantly decreased as compared with wild-type bacteria. In a murine pneumonia model, wild-type bacteria exhibited a severe lung pathology and induced a high bacterial burden in blood, whereas AbOmpA- mutant was rarely detected in blood. Conclusion A. baumannii adheres to and invades epithelial cells. AbOmpA plays a major role in the interactions with epithelial cells. These findings contribute to the understanding of A. baumannii pathogenesis in the early stage of bacterial infection.

  3. Gefarnate stimulates mucin-like glycoprotein secretion in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models

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    Dota A

    2013-01-01

    Full Text Available Atsuyoshi Dota, Yuko Takaoka-Shichijo, Masatsugu NakamuraOphthalmic Research and Development Center, Santen Pharmaceutical Co, Ltd, Ikoma-shi, Nara, JapanPurpose: The aim of this study was to evaluate the effect of gefarnate on mucin-like glycoprotein secretion in isolated rabbit conjunctival tissue, and on corneal epithelial damage in rabbit and cat dry-eye models.Methods: Conjunctival tissue isolated from rabbits was treated with gefarnate. Mucin-like glycoprotein was detected in the culture supernatant by an enzyme-linked lectin assay. Gefarnate ointment was topically applied to eyes once daily for 7 days in the rabbit dry-eye model, in which the lacrimal glands, Harderian gland, and nictitating membrane were removed, or for 4 weeks in the cat dry-eye model, in which the lacrimal gland and nictitating membrane were removed. Corneal epithelial damage was evaluated by measurement of corneal permeability by rose bengal in the rabbit model or by fluorescein staining in the cat model.Results: Gefarnate stimulated mucin-like glycoprotein secretion in conjunctival tissue in a dose-dependent manner. In the rabbit dry-eye model, application of gefarnate ointment to the eyes resulted in a dose-dependent decrease in rose bengal permeability in the cornea, with the effect being significant at concentrations of ≥0.3%. In the cat dry-eye model, application of gefarnate ointment resulted in a significant decrease in the corneal fluorescein staining score.Conclusion: These results suggest that gefarnate stimulates in vitro secretion of mucin-like glycoprotein in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models. Gefarnate may therefore be effective for treating dry eye.Keywords: gefarnate, fluorescein staining, rose bengal permeability, rabbit, cat, dry eye

  4. Effect of rigid corneal contact lens and corneal limbal stem cell transplantation for senile patients with pterygium

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    Jiang Lu

    2017-06-01

    Full Text Available AIM: To investigate the effect of rigid contact lens in the treatment after pterygium excision and limbal stem cell transplantation in senile patients. METHODS: Totally 90 elderly patients diagnosed as unilateral pterygium in our hospital from March 2015 to March 2016 were selected and divided into two groups, observation group and control group, 45 case with 45 eyes in each group. Observation group was treated with limbal stem cell transplantation and rigid contact lens. Control group was treated with limbal stem cell transplantation only. The following indicators were observed and compared: corneal healing time, visual analogue score(VASat 1, 3, 5 and 7d after surgery and the recurrence rate of pterygium. RESULTS: The score on corneal irritation of observation group was significantly lower than that of control group(PPPP>0.05.CONCLUSION: Autologous corneal stem cell transplantation combined with rigid contact lens for pterygium in elderly patients is effective with shorter healing time and less pain, and it does not increase the recurrence rate.

  5. Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC as a cellular alternative for in vitro ocular toxicity testing.

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    Edith Aberdam

    Full Text Available Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

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

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    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.

  7. Feasibility of cell-based therapy combined with descemetorhexis for treating Fuchs endothelial corneal dystrophy in rabbit model.

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    Naoki Okumura

    Full Text Available Corneal transparency is maintained by the corneal endothelium through its pump and barrier function. Severe corneal endothelial damage results in dysregulation of water flow and eventually causes corneal haziness and deterioration of visual function. In 2013, we initiated clinical research of cell-based therapy for treating corneal decompensation. In that study, we removed an 8-mm diameter section of damaged corneal endothelium without removing Descemet's membrane (the basement membrane of the corneal endothelium and then injected cultured human corneal endothelial cells (CECs into the anterior chamber. However, Descemet's membrane exhibits clinically abnormal structural features [i.e., multiple collagenous excrescences (guttae and thickening] in patients with Fuchs endothelial corneal dystrophy (FECD and the advanced cornea guttae adversely affects the quality of vision, even in patients without corneal edema. The turnover time of cornea guttae is also not certain. Therefore, we used a rabbit model to evaluate the feasibility of Descemet's membrane removal in the optical zone only, by performing a small 4-mm diameter descemetorhexis prior to CEC injection. We showed that the corneal endothelium is regenerated both on the corneal stroma (the area of Descemet's membrane removal and on the intact peripheral Descemet's membrane, based on the expression of function-related markers and the restoration of corneal transparency. Recovery of the corneal transparency and central corneal thickness was delayed in areas of Descemet's membrane removal, but the cell density of the regenerated corneal endothelium and the thickness of the central corneal did not differ between the areas with and without residual Descemet's membrane at 14 days after CEC injection. Here, we demonstrate that removal of a pathological Descemet's membrane by a small descemetorhexis is a feasible procedure for use in combination with cell-based therapy. The current strategy might be

  8. Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation

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    Li Cui

    2014-06-01

    Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.

  9. Optimization of immunolocalization of cell cycle proteins in human corneal endothelial cells

    Science.gov (United States)

    He, Zhiguo; Campolmi, Nelly; Ha Thi, Binh-Minh; Dumollard, Jean-Marc; Peoc’h, Michel; Garraud, Olivier; Piselli, Simone; Gain, Philippe

    2011-01-01

    Purpose En face observation of corneal endothelial cells (ECs) using flat-mounted whole corneas is theoretically much more informative than observation of cross-sections that show only a few cells. Nevertheless, it is not widespread for immunolocalization (IL) of proteins, probably because the endothelium, a superficial monolayer, behaves neither like a tissue in immunohistochemistry (IHC) nor like a cell culture in immunocytochemistry (ICC). In our study we optimized IL for ECs of flat-mounted human corneas to study the expression of cell cycle-related proteins. Methods We systematically screened 15 fixation and five antigen retrieval (AR) methods on 118 human fresh or stored corneas (organ culture at 31 °C), followed by conventional immunofluorescence labeling. First, in an attempt to define a universal protocol, we selected combinations able to correctly localize four proteins that are perfectly defined in ECs (zonula occludens-1 [ZO-1] and actin) or ubiquitous (heterogeneous nuclear ribonucleoprotein L [hnRNP L] and histone H3). Second, we screened protocols adapted to the revelation of 9 cell cycle proteins: Ki67, proliferating cell nuclear antigen (PCNA), minichromosome maintenance protein 2 (MCM2), cyclin D1, cyclin E, cyclin A, p16Ink4a, p21Cip1 and p27Kip1. Primary antibody controls (positive controls) were performed on both epithelial cells of the same, simultaneously-stained whole corneas, and by ICC on human ECs in in vitro non-confluent cultures. Both controls are known to contain proliferating cells. IL efficiency was evaluated by two observers in a masked fashion. Correct localization at optical microscopy level in ECs was define as clear labeling with no background, homogeneous staining, agreement with previous works on ECs and/or protein functions, as well as a meaningful IL in proliferating cells of both controls. Results The common fixation with 4% formaldehyde (gold standard for IHC) failed to reveal 12 of the 13 proteins. In contrast, they

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

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    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.

  11. Early Intervention Stem Cell-Based Therapy (EISCBT) for Corneal Burns and Trauma

    Science.gov (United States)

    2016-10-01

    AWARD NUMBER: W81XWH-14-1-0465 TITLE: Early Intervention Stem Cell -Based Therapy (EISCBT) for Corneal Burns and Trauma PRINCIPAL INVESTIGATOR...Intervention Stem Cell -Based Therapy (EISCBT) for Corneal Burns and Trauma 5a. CONTRACT NUMBER W81WH-14-1-0465 5b. GRANT NUMBER 5c. PROGRAM...Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The goal of this project is to develop a stem cell -based regenerative

  12. Development of Thymic Epithelial Cells

    DEFF Research Database (Denmark)

    Ulyanchenko, Svetlana; Vaidya, Harsh J.; O'Neill, Kathy E.

    2016-01-01

    The thymus is the primary lymphoid organ in which the T cell repertoire is generated. The complex cellularity of this organ is uniquely designed to facilitate T cell development: defects in thymus development or function can cause immunodeficiencies ranging from the absence of T cell-mediated imm......The thymus is the primary lymphoid organ in which the T cell repertoire is generated. The complex cellularity of this organ is uniquely designed to facilitate T cell development: defects in thymus development or function can cause immunodeficiencies ranging from the absence of T cell......-mediated immunity to broad-spectrum autoimmune disease. Peak thymus size and output occurs early in life, after which the thymus undergoes a natural process of involution. This results in the progressive loss of functional thymus tissue and correspondingly in decreased production of new naïve T cells with age...... - contributing to the diminished capacity of the aged immune system to adequately respond to new antigenic challenge. Age-related thymic involutions, together with the thymic involutions associated with cytotoxic therapies (e.g., radio- or chemotherapy), have raised interest in development of clinically useful...

  13. 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.

  14. Mutations in TCF8 Cause Posterior Polymorphous Corneal Dystrophy and Ectopic Expression of COL4A3 by Corneal Endothelial Cells

    Science.gov (United States)

    Krafchak, Charles M. ; Pawar, Hemant ; Moroi, Sayoko E. ; Sugar, Alan ; Lichter, Paul R. ; Mackey, David A. ; Mian, Shahzad ; Nairus, Theresa ; Elner, Victor ; Schteingart, Miriam T. ; Downs, Catherine A. ; Kijek, Theresa Guckian ; Johnson, Jenae M. ; Trager, Edward H. ; Rozsa, Frank W. ; Ali Mandal, Md Nawajes ; Epstein, Michael P. ; Vollrath, Douglas ; Ayyagari, Radha ; Boehnke, Michael ; Richards, Julia E. 

    2005-01-01

    Posterior polymorphous corneal dystrophy (PPCD, also known as PPMD) is a rare disease involving metaplasia and overgrowth of corneal endothelial cells. In patients with PPCD, these cells manifest in an epithelial morphology and gene expression pattern, produce an aberrant basement membrane, and, sometimes, spread over the iris and nearby structures in a way that increases the risk for glaucoma. We previously mapped PPCD to a region (PPCD3) on chromosome 10 containing the gene that encodes the two-handed zinc-finger homeodomain transcription factor TCF8. Here, we report a heterozygous frameshift mutation in TCF8 that segregates with PPCD in the family used to map PPCD3 and four different heterozygous nonsense and frameshift mutations in TCF8 in four other PPCD probands. Family reports of inguinal hernia, hydrocele, and possible bone anomalies in affected individuals suggest that individuals with TCF8 mutations should be examined for nonocular anomalies. We detect transcripts of all three identified PPCD genes (VSX1, COL8A2, and TCF8) in the cornea. We show presence of a complex (core plus secondary) binding site for TCF8 in the promoter of Alport syndrome gene COL4A3, which encodes collagen type IV α3, and we present immunohistochemical evidence of ectopic expression of COL4A3 in corneal endothelium of the proband of the original PPCD3 family. Identification of TCF8 as the PPCD3 gene provides a valuable tool for the study of critical gene regulation events in PPCD pathology and suggests a possible role for TCF8 mutations in altered structure and function of cells lining body cavities other than the anterior chamber of the eye. Thus, this study has identified TCF8 as the gene responsible for approximately half of the cases of PPCD, has implicated TCF8 mutations in developmental abnormalities outside the eye, and has presented the TCF8 regulatory target, COL4A3, as a key, shared molecular component of two different diseases, PPCD and Alport syndrome. PMID:16252232

  15. Traction forces exerted by epithelial cell sheets

    International Nuclear Information System (INIS)

    Saez, A; Anon, E; Ghibaudo, M; Di Meglio, J-M; Hersen, P; Ladoux, B; Du Roure, O; Silberzan, P; Buguin, A

    2010-01-01

    Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

  16. 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

  17. Generation of Mouse Lung Epithelial Cells.

    Science.gov (United States)

    Kasinski, Andrea L; Slack, Frank J

    2013-08-05

    Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of Kras LSL-G12D/+ ; p53 LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra -G12D and p53 R172 . While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

  18. Multivariate analysis of corneal endothelial cell count reduction after cataract surgery

    Directory of Open Access Journals (Sweden)

    Duan-Rong Cao

    2018-04-01

    Full Text Available AIM:To investigate the factors related to the decrease of corneal endothelial cell number after phacoemulsification in cataract patients. METHODS: We selected 98 patients(120 eyesin Ophthalmic Center from July 2014 to July 2016 underwent phacoemulsification and they were retrospectively analyzed. According to the central corneal endothelial cell density before and 2mo after the operation, they were divided into serious loss group of 52 cases(67 eyes, density of central corneal endothelial cells loss rate no less than 12.3%, the general loss group of 46 cases(53 eyes, the density of central corneal endothelial cell loss rate RESULTS: Serious loss group and the general group on gender, rate with hypertension, rate with diabetes, rate with high blood lipids, with shallow anterior chamber, corneal diameter and suction time comparison, had no statistically significant differences(P>0.05. Nuclear hardness classification of Emery lens, ultrasonic power, ultrasonic emulsification time, age between groups were significantly different(PPCONCLUSION: The main factors that influence the decrease of corneal endothelial cell number after phacoemulsification are Emery lens, higher grade of nucleus of lens, increase of ultrasonic energy, longer time of phacoemulsification and increased age.

  19. Corneal Laceration

    Medline Plus

    Full Text Available ... What Is Corneal Laceration? Corneal Laceration Symptoms What Causes Corneal Laceration? Corneal Laceration Diagnosis Corneal Laceration Treatment ... the corneal laceration is deep enough it can cause a full thickness laceration. This is when the ...

  20. Persistent Epithelial Defects and Corneal Opacity After Collagen Cross-Linking With Substitution of Dextran (T-500) With Dextran Sulfate in Compounded Topical Riboflavin.

    Science.gov (United States)

    Höllhumer, Roland; Watson, Stephanie; Beckingsale, Peter

    2017-03-01

    Collagen cross-linking (CXL) is a commonly performed procedure to prevent the progression of keratoconus. Riboflavin is an essential part of the procedure, which facilitates both the cross-linking process and protection of intraocular structures. Dextran can be added to riboflavin to create an isotonic solution. This case report highlights the importance of compounding riboflavin with the correct dextran solution. A retrospective case series. Six eyes of 4 male patients with keratoconus aged from 20 to 38 years underwent CXL with substitution of 20% dextran (T-500) with 20% dextran sulfate in a compounded riboflavin 0.1% solution. Postoperatively, persistent corneal epithelial defects, stromal haze, and then scarring occurred. Corneal transplantation was performed for visual rehabilitation but was complicated by graft rejection followed by failure (n = 1 eye), dehiscence (n = 4), cataract (n = 2), post-laser ablation haze (n = 1), and steroid-induced glaucoma (n = 2). The visual outcome was riboflavin solutions during CXL results in loss of vision from permanent corneal opacity. Residual host changes may compromise the results of corneal transplantation.

  1. 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.

  2. [Cell-based therapies - an innovative therapeutic option in ophthalmology: Treating corneal diseases with stem cells].

    Science.gov (United States)

    Bakker, Ann-Christin; Langer, Barbara

    2015-11-01

    Pathological changes and disorders of the cornea are a major cause of severe visual impairment and blindness. Replacement of a pathologically altered cornea with healthy corneal tissue from the eye of a suitable donor is among the most common and successful transplantation procedures in medicine. In Germany, approximately 5000-6000 corneal transplantations are performed each year, but the total demand per year is estimated to be twice as high. With a success rate of 90%, the outcome of cornea transplantation is very favourable. However, long-term maintenance and regeneration of a healthy new cornea requires tissue-specific corneal stem cells residing at the basal layer of the limbus, which is the annular transition zone between the cornea and sclera. When this important limbal stem cell population is destroyed or dysfunctional, a pathological condition known as limbal stem cell deficiency (LSCD) manifests. Limbal stem cell deficiency describes conditions associated with impaired corneal wound healing and regeneration. In this situation, transplantation of healthy limbal stem cells is the only curative treatment approach for restoration of an intact and functional ocular surface. To date, treatment of LSCD presents a great challenge for ophthalmologists. However, innovative, cell-therapeutic approaches may open new, promising treatment perspectives. In February 2015, the European Commission granted marketing authorization to the first stem cell-based treatment in the European Union. The product named Holoclar® is an advanced therapy medicinal product (ATMP) for the treatment of moderate to severe LSCD due to physical and chemical burns in adults. Further cell-based treatment approaches are in clinical development.

  3. 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....

  4. 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.

  5. Electrical estimulation of retinal pigment epithelial cells.

    Science.gov (United States)

    Gamboa, Olga Lucia; Pu, Jin; Townend, John; Forrester, John V; Zhao, Min; McCaig, Colin; Lois, Noemi

    2010-08-01

    We investigated and characterized the effect of externally applied electric fields (EF) on retinal pigment epithelial (RPE) cells by exposing primary cultures of human RPE cells (hRPE) and those from the ARPE19 immortalized cell line to various strengths of EF (EF-treated cells) or to no EF (control cells) under different conditions including presence or absence of serum and gelatin and following wounding. We evaluated changes in RPE cell behavior in response to EF by using a computer based image capture and analysis system (Metamorph). We found that RPE cells responded to externally applied EFs by preferential orientation perpendicular to the EF vector, directed migration towards the anode, and faster translocation rate than control, untreated cells. These responses were voltage-dependent. Responses were observed even at low voltages, of 50-300 mV. Furthermore, the migration of hRPE cell sheets generated by wounding of confluent monolayers of cells at early and late confluence could be manipulated by the application of EF, with directed migration towards the anode observed at both sides of the wounded hRPE. In conclusion, RPE cell behaviour can be controlled by an externally applied EF. The potential for externally applied EF to be used as a therapeutic strategy in the management of selected retinal diseases warrants further investigation. Copyright 2010 Elsevier Ltd. All rights reserved.

  6. Hyaluronan protection of corneal endothelial cells against extracellular histones after phacoemulsification.

    Science.gov (United States)

    Kawano, Hiroki; Sakamoto, Taiji; Ito, Takashi; Miyata, Kazunori; Hashiguchi, Teruto; Maruyama, Ikuro

    2014-11-01

    To determine the effect of histones on corneal endothelial cells generated during cataract surgery. Kagoshima University Hospital, Kagoshima, Japan. Experimental study. Standard phacoemulsification was performed on enucleated pig eyes. Histones in the anterior segment of the eye were determined by immunohistochemistry. Cultured human corneal endothelial cells were exposed to histones for 18 hours, and cell viability was determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay. The concentration of interleukin-6 (IL-6) in the culture medium of human corneal endothelial cells was measured using enzyme-linked immunosorbent assay. The effects of signal inhibitors U0126, SB203580, and SP600125 were evaluated. The protective effect of hyaluronan against histones was evaluated in human corneal endothelial cells with and without hyaluronan. Cellular debris containing histones was observed in the anterior chamber of pig eyes after phacoemulsification. Exposure of human corneal endothelial cells to 50 μg/mL of histones or more led to cytotoxic effects. The IL-6 concentration was significantly increased dose dependently after exposure of human corneal endothelial cells to histones (Phistone-induced IL-6 production was significantly decreased by extracellular signal-regulated kinases 1/2 and p-38 mitogen-activated protein kinase inhibitors (Phistones caused formation of histone aggregates, decreased the cytotoxic effects of the histones, and blocked the increase in IL-6 (PHistones were released extracellularly during phacoemulsification and exposure of human corneal endothelial cells to histones increased the IL-6 secretion. The intraoperative use of hyaluronan may decrease the cytotoxic effects of histones released during cataract surgery. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  7. 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.

  8. 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.

  9. Current perspectives in epithelial cell injury and repair: consequences for epithelial functions

    Directory of Open Access Journals (Sweden)

    R. Lutter

    2005-12-01

    Full Text Available Epithelial cells lining the airways and the respiratory compartment may, and certainly when exposed to an inflammatory milieu, display an altered functioning, which could contribute to pathophysiology of inflammatory lung/airway disease. In the present review paper, several issues that were discussed at an earlier European Respiratory Society Research Seminar on conditions that affect epithelial functioning have been recapitulated and updated. These and future studies should improve understanding of epithelial functioning and may aid recovery from disease.

  10. Novel Application of Artificial Dermis Plus Autologous Vital Epithelial Cells: Improved Wound Epithelialization

    Directory of Open Access Journals (Sweden)

    Li-Tzu Lee

    2010-02-01

    Full Text Available The purpose of this study was to evaluate artificial dermis with the simultaneous addition of autologous epithelial cells for oral lesion defect reconstruction. Surgical wounds reconstructed with artificial dermis plus scraped epithelial cells were evaluated in 5 patients with oral benign lesions or squamous cell carcinoma. Clinical follow-up indices included scar formation and tissue surface texture observation. The neomucosal layers were analyzed histologically to establish the degree of epithelialization. Clinical observation showed that the oral mucosal texture was smoother in artificial dermis with added epithelial cells at 4 weeks postoperation compared with artificial dermis alone. The wound contraction and scar formation processes were slow. Viable epithelial cells with flat rete ridges remained in the artificial dermis, and a neoepithelial layer was present in the histological findings. We showed that healthy granulation tissue and neoepithelial formation in artificial dermis with epithelial cells was beneficial for the repair of oral defects. Scraping oral epithelial cells and applying them to artificial dermis assisted in the early preparation of composite grafts and minimized requirement for donor sites. This technique may improve the treatment of patients with oral benign tumors and early-stage squamous cell carcinoma.

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

    Directory of Open Access Journals (Sweden)

    Liang Chen

    2011-01-01

    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.

  12. Gene expressions changes in bronchial epithelial cells

    DEFF Research Database (Denmark)

    Remy, S.; Verstraelen, S.; Van Den Heuvel, R.

    2014-01-01

    For the classification of respiratory sensitizing chemicals, no validated in vivo nor in vitro tests are currently available. In this study, we evaluated whether respiratory sensitizers trigger specific signals in human bronchial epithelial (BEAS-2B) cells at the level of the transcriptome...... oligonucleotide arrays. A limited number of 11 transcripts could be identified as potential biomarkers to identify respiratory sensitizers. Three of these transcripts are associated to immune system processes (HSPA5, UPP1, and SEPRINEI). In addition, the transcriptome was screened for transcripts....... The cells were exposed during 6, 10, and 24 h to 4 respiratory sensitizers and 6 non-respiratory sensitizers (3 skin sensitizers and 3 respiratory irritants) at a concentration inducing 20% cell viability loss after 24 h. Changes in gene expression were evaluated using Agilent Whole Human Genome 4 x 44 K...

  13. Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.

    Science.gov (United States)

    Hamuro, Junji; Toda, Munetoyo; Asada, Kazuko; Hiraga, Asako; Schlötzer-Schrehardt, Ursula; Montoya, Monty; Sotozono, Chie; Ueno, Morio; Kinoshita, Shigeru

    2016-09-01

    To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 μm2, and the density of cHCECs exceeded 2500 cells/mm2. A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

  14. Caveolin-1 as a Novel Indicator of Wound-Healing Capacity in Aged Human Corneal Epithelium

    OpenAIRE

    Rhim, Ji Heon; Kim, Jae Hoon; Yeo, Eui-Ju; Kim, Jae Chan; Park, Sang Chul

    2010-01-01

    Excess caveolin-1 has been reported to play a role in age-dependent hyporesponsiveness to growth factors in vitro. Therefore, we hypothesized that caveolin-1–dependent hyporesponsiveness to growth factors in aged corneal epithelial cells might be responsible for delayed wound healing in vivo. To test this hypothesis, we evaluated corneal wound-healing time by vital staining using fluorescein after laser epithelial keratomileusis (LASEK). We compared wound-healing times in young, middle-aged a...

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Cellular Plasticity of Epithelial Cells-Cause of Metastasis

    National Research Council Canada - National Science Library

    Sukumar, Saraswati

    2005-01-01

    .... We present a novel concept that cancer epithelial cells, possibly of stem cell origin, have inherent cellular plasticity and can differentiate into endothelial cells and form microvessels that serve...

  20. Generation and Feasibility Assessment of a New Vehicle for Cell-Based Therapy for Treating Corneal Endothelial Dysfunction.

    Directory of Open Access Journals (Sweden)

    Naoki Okumura

    Full Text Available The corneal endothelium maintains corneal transparency by its pump and barrier functions; consequently, its decompensation due to any pathological reason causes severe vision loss due to corneal haziness. Corneal transplantation is the only therapeutic choice for treating corneal endothelial dysfunction, but associated problems, such as a shortages of donor corneas, the difficulty of the surgical procedure, and graft failure, still need to be resolved. Regenerative medicine is attractive to researchers as a means of providing innovative therapies for corneal endothelial dysfunction, as it now does for other diseases. We previously demonstrated the successful regeneration of corneal endothelium in animal models by injecting cultured corneal endothelial cells (CECs in combination with a Rho kinase (ROCK inhibitor. The purpose of the present study was to optimize the vehicle for clinical use in cell-based therapy. Our screening of cell culture media revealed that RELAR medium promoted CEC adhesion. We then modified RELAR medium by removing hormones, growth factors, and potentially toxic materials to generate a cell therapy vehicle (CTV composed of amino acid, salts, glucose, and vitamins. Injection of CECs in CTV enabled efficient engraftment and regeneration of the corneal endothelium in the rabbit corneal endothelial dysfunction model, with restoration of a transparent cornea. The CECs retained >85% viability after a 24 hour preservation as a cell suspension in CTV at 4°C and maintained their potency to regenerate the corneal endothelium in vivo. The vehicle developed here is clinically applicable for cell-based therapy aimed at treating the corneal endothelium. Our strategy involves the generation of vehicle from a culture medium appropriate for a given cell type by removing materials that are not favorable for clinical use.

  1. Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium.

    Directory of Open Access Journals (Sweden)

    Makiko Nakahara

    Full Text Available Healthy corneal endothelium is essential for maintaining corneal clarity, as the damage of corneal endothelial cells and loss of cell count causes severe visual impairment. Corneal transplantation is currently the only therapy for severe corneal disorders. The greatly limited proliferative ability of human corneal endothelial cells (HCECs, even in vitro, has challenged researchers to establish efficient techniques for the cultivating HCECs, a pivotal issue for clinical applications. The aim of this study was to evaluate conditioned medium (CM obtained from human bone marrow-derived mesenchymal stem cells (MSCs (MSC-CM for use as a consistent expansion protocol of HCECs. When HCECs were maintained in the presence of MSC-CM, cell morphology assumed a hexagonal shape similar to corneal endothelial cells in vivo, as opposed to the irregular cell shape observed in control cultures in the absence of MSC-CM. They also maintained the functional protein phenotypes; ZO-1 and Na(+/K(+-ATPase were localized at the intercellular adherent junctions and pump proteins of corneal endothelium were accordingly expressed. In comparison to the proliferative potential observed in the control cultures, HCECs maintained in MSC-CM were found to have more than twice as many Ki67-positive cells and a greatly increased incorporation of BrdU into DNA. MSC-CM further facilitated the cell migration of HCECs. Lastly, the mechanism of cell proliferation mediated by MSC-CM was investigated, and phosphorylation of Akt and ERK1/2 was observed in HCECs after exposure to MSC-CM. The inhibitor to PI 3-kinase maintained the level of p27(Kip1 for up to 24 hours and greatly blocked the expression of cyclin D1 and D3 during the early G1 phase, leading to the reduction of cell density. These findings indicate that MSC-CM not only stimulates the proliferation of HCECs by regulating the G1 proteins of the cell cycle but also maintains the characteristic differentiated phenotypes necessary

  2. Effects of intraocular irrigating solutions on the spreading of rabbit corneal endothelial cells on extracellular matrices.

    Science.gov (United States)

    Nishida, T; Otori, T

    1991-01-01

    The effects of four commercially available irrigating solutions on the spreading of rabbit corneal endothelial cells on various extracellular matrices were studied. Cultured rabbit corneal endothelial cells, suspended in one of the following intraocular irrigating solutions, Opeguard MA, BSS, BSS Plus, lactated Ringer solution (Lactec) or physiological saline, were placed on uncoated tissue culture plates or on plates coated with extracellular matrices (fibronectin, laminin, collagen type I, or collagen type IV). The cell area was measured after 45 minutes' incubation. The cells spread on all of the extracellular matrices examined but not on the uncoated tissue culture plates. On the fibronectin or laminin matrix, the cell area was significantly greater with Opeguard MA or BSS Plus. On laminin and collagen type IV, the cell area was the greatest with Opeguard MA. On collagen type I, the cell area was significantly greater with Opeguard MA, BSS, or BSS Plus. These results demonstrated that the rabbit corneal endothelial cells responded to the extracellular matrices, and that Opeguard MA or BSS Plus provided more favorable conditions for the spreading of these cells. These results indicated that both Opeguard MA and BSS Plus might aid the spreading of corneal endothelial cells during wound-healing immediately after intraocular surgery.

  3. Arachidonic acid promotes epithelial-to-mesenchymal-like transition in mammary epithelial cells MCF10A.

    Science.gov (United States)

    Martinez-Orozco, Raul; Navarro-Tito, Napoleon; Soto-Guzman, Adriana; Castro-Sanchez, Luis; Perez Salazar, Eduardo

    2010-06-01

    Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of breast cancer. Cancer progression requires the development of metastasis, which is characterized by an increase in cell motility and invasion. Epithelial-to-mesenchymal transition (EMT) is a process, by which epithelial cells are transdifferentiated to a more mesenchymal state. A similar process takes place during tumor progression, when carcinoma cells stably or transiently lose epithelial polarities and acquire a mesenchymal phenotype. Arachidonic acid (AA) is a fatty acid that mediates cellular processes, such as cell survival, angiogenesis, chemotaxis, mitogenesis, migration and apoptosis. However, the role of AA on the EMT process in human mammary epithelial cells remains to be studied. We demonstrate here that AA promotes an increase in vimentin and N-cadherin expression, MMP-9 secretion, a decrease in E-cadherin junctional levels, and the activation of FAK, Src and NF-kappaB in MCF10A cells. Furthermore, AA also promotes cell migration in an Src kinase activity-dependent fashion. In conclusion, our results demonstrate, for the first time, that AA promotes an epithelial-to-mesenchymal-like transition in MCF10A human mammary non-tumorigenic epithelial cells. 2010 Elsevier GmbH. All rights reserved.

  4. Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

    Directory of Open Access Journals (Sweden)

    Hannah J Levis

    Full Text Available Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

  5. induced acute cytotoxicity in human cervical epithelial carcinoma cells

    African Journals Online (AJOL)

    Molecular basis of arsenite (As +3 )-induced acute cytotoxicity in human cervical epithelial carcinoma cells. ... Libyan Journal of Medicine ... Methods: After performing cytotoxic assays on a human epithelial carcinoma cell line, expression analysis was done by quantitative polymerase chain reaction, western blotting, and ...

  6. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    This study aims to investigate EPCR expression in renal tubular epithelial cells and related influencing factors. EPCR expression was assessed by flow cytometry in renal tubular epithelial cells. The effects of some reagents (high glucose, tumor necrosis factor–α and interleukin-1β) were measured by RT-PCR. The results ...

  7. Oxidant-mediated epithelial cell injury in idiopathic pulmonary fibrosis.

    OpenAIRE

    Cantin, A M; North, S L; Fells, G A; Hubbard, R C; Crystal, R G

    1987-01-01

    Lung inflammatory cells of patients with idiopathic pulmonary fibrosis (IPF) were evaluated for their ability to injure 51Cr-labeled AKD alveolar epithelial cells in the presence and absence of IPF alveolar epithelial lining fluid (ELF). The IPF cells were spontaneously releasing exaggerated amounts of superoxide (O.2) and hydrogen peroxide (H2O2) compared with normal (P less than 0.02). Cytotoxicity of the AKD cells was markedly increased when the IPF inflammatory cells were incubated with a...

  8. The effect of intravitreal bevacizumab injection on the corneal endothelial cells

    Directory of Open Access Journals (Sweden)

    Akbar Derakhshan

    2016-04-01

    Full Text Available Introduction:Bevacizumab (Avastin, as an effectiveness treatment modality, is currently used in patients with various ocular disease. However the results have been promising, the use of bevacizumab in the treatment of ocular disease is an off-label application. Hence, the aim of this study was to systematically review the effectiveness of intravitreal injection of bevacizumab on various ocular tissues, especially corneal endothelial cells. Methods: The articles related to the effect of application of Avastin in the treatment of ophthalmic diseases and especially its effect on corneal endothelial cells were collected and reviewed. We searched PubMed, Google scholar, and Scopus databases and used Avastin, ocular diseases and corneal endothelial cells as search keywords.Result: Of all 55 articles found in all databases, only 10 were relevant to the purpose of this study, and 45 articles were excluded in several step by step process of article selection according to the inclusion/exclusion criteria. The results revealed that intracameral bevacizumab injection caused no changes in specular microscopy and corneal pachymetry. Moreover, it had no significant toxicity on corneal endothelial cells.Discussion: Effectiveness of bevacizumab as a new modality in the treatment of different ophthalmic diseases have been suggested. Recent data on both human and animal models showed that intravitreal injection of bevacizumab resulted in no significant toxicity on various ocular cells, and it could be considered as a suitable therapeutic approach in clinical use.Conclusion: According to the results of included documents, bevacizumab was not toxic to corneal endothelial cells at various clinically relevant doses.

  9. 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.

  10. Establishment of functioning human corneal endothelial cell line with high growth potential.

    Directory of Open Access Journals (Sweden)

    Tadashi Yokoi

    Full Text Available Hexagonal-shaped human corneal endothelial cells (HCEC form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na(+- and K(+-dependent ATPase (Na(+/K(+-ATPase. Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs in the Rb pathway (p16-CDK4/CyclinD1-pRb. In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7 and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin. Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7, THCEH (Cyclin and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7 and THCEH (Cyclin. THCEH (Cyclin expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na(+/K(+-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7. This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology.

  11. Multi-functionality and plasticity characterize epithelial cells in Hydra

    Science.gov (United States)

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  12. A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

    Science.gov (United States)

    Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

    2018-01-01

    Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

  13. 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.

  14. Cytomatrix synthesis in MDCK epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, J.J.; Low, R.B.; Woodcock-Mitchell, J.L. (Univ. of Vermont, Burlington (USA))

    1990-06-01

    Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak, was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with (14C)leucine over several days and then pulse-labeled for 4 hours with (3H)leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form.

  15. 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.

  16. 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

  17. Transplantation of retinal pigment epithelial cells - a possible future treatment for age-related macular degeneration

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

    ophthalmology, age-related macular degeneration, retinal pigment epithelial cells, transplantation, treatment......ophthalmology, age-related macular degeneration, retinal pigment epithelial cells, transplantation, treatment...

  18. Transplantation of retinal pigment epithelial cells - a possible future treatment for age-related macular degeneration

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

    ophthalmology, age-related macular degeneration, transplantation, retinal pigment epithelial cells, treatment......ophthalmology, age-related macular degeneration, transplantation, retinal pigment epithelial cells, treatment...

  19. Streptococcus equi subsp zooepidemicus Invades and Survives in Epithelial Cells

    DEFF Research Database (Denmark)

    Skive, Bolette; Rohde, Manfred; Molinari, Gabriella

    2017-01-01

    showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells...... protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive...

  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. Mesenchymal-­epithelial interactions during digestive tract development and epithelial stem cell regeneration

    Science.gov (United States)

    Le Guen, Ludovic; Marchal, Stéphane; Faure, Sandrine; De Santa Barbara, Pascal

    2015-01-01

    The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal–epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration. PMID:26126787

  2. 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

  3. Epithelial-mesenchymal transition in breast epithelial cells treated with cadmium and the role of Snail.

    Science.gov (United States)

    Wei, Zhengxi; Shan, Zhongguo; Shaikh, Zahir A

    2018-04-01

    Epidemiological and experimental studies have implicated cadmium (Cd) with breast cancer. In breast epithelial MCF10A and MDA-MB-231 cells, Cd has been shown to promote cell growth. The present study examined whether Cd also promotes epithelial-mesenchymal transition (EMT), a hallmark of cancer progression. Human breast epithelial cells consisting of non-cancerous MCF10A, non-metastatic HCC 1937 and HCC 38, and metastatic MDA-MB-231 were treated with 1 or 3 μM Cd for 4 weeks. The MCF10A epithelial cells switched to a more mesenchymal-like morphology, which was accompanied by a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal markers N-cadherin and vimentin. In both non-metastatic HCC 1937 and HCC 38 cells, treatment with Cd decreased the epithelial marker claudin-1. In addition, E-cadherin also decreased in the HCC 1937 cells. Even the mesenchymal-like MDA-MB-231 cells exhibited an increase in the mesenchymal marker vimentin. These changes indicated that prolonged treatment with Cd resulted in EMT in both normal and cancer-derived breast epithelial cells. Furthermore, both the MCF10A and MDA-MB-231 cells labeled with Zcad, a dual sensor for tracking EMT, demonstrated a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker ZEB-1. Treatment of cells with Cd significantly increased the level of Snail, a transcription factor involved in the regulation of EMT. However, the Cd-induced Snail expression was completely abolished by actinomycin D. Luciferase reporter assay indicated that the expression of Snail was regulated by Cd at the promotor level. Snail was essential for Cd-induced promotion of EMT in the MDA-MB-231 cells, as knockdown of Snail expression blocked Cd-induced cell migration. Together, these results indicate that Cd promotes EMT in breast epithelial cells and does so by modulating the transcription of Snail. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Corneal endothelial cell loss and corneal biomechanical characteristics after two-step sequential or combined phaco-vitrectomy surgery for idiopathic epiretinal membrane

    DEFF Research Database (Denmark)

    Hamoudi, Hassan; Christensen, Ulrik Correll; La Cour, Morten

    2017-01-01

    allocated to (i) cataract surgery and subsequent PPV (CAT group), (ii) PPV and subsequent cataract surgery (VIT group) or (iii) phacovitrectomy (COMBI group). Eyes were examined at baseline, 1 month after each surgery, and at 3 and 12 months follow-up. Corneal endothelium cell density (CED) was assessed......PURPOSE: To assess the impact of sequential and combined surgery [cataract surgery and 23-gauge pars plana vitrectomy (PPV) with peeling] on corneal endothelium cell density (CED) and corneal biomechanical characteristics. METHODS: Phakic eyes with epiretinal membrane (ERM) were prospectively...... with non-contact specular microscopy. Pachymetry [central cornea thickness (CCT)], keratometry and cornea volume (CV) were measured with Pentacam Scheimpflug camera. Primary outcome was change in CED after 12 months; secondary outcomes were changes in CCT and CV after 12 months. RESULTS: Sixty-two eyes...

  5. Hormonal regulation of Na+/K+-dependent ATPase activity and pump function in corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin

    2011-10-01

    Na- and K-dependent ATPase (Na,K-ATPase) in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in these cells. Mouse corneal endothelial cells were exposed to dexamethasone or insulin. ATPase activity was evaluated by spectrophotometric measurement, and pump function was measured using an Ussing chamber. Western blotting and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. Dexamethasone increased Na,K-ATPase activity and the pump function of endothelial cells. Western blot analysis indicated that dexamethasone increased the expression of the Na,K-ATPase α1-subunit but decreased the ratio of active to inactive Na,K-ATPase α1-subunit. Insulin increased Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were transient and blocked by protein kinase C inhibitors and inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A). Western blot analysis indicated that insulin decreased the amount of inactive Na,K-ATPase α1-subunit, but the expression of total Na,K-ATPase α1-subunit was unchanged. Immunocytochemistry showed that insulin increased cell surface expression of the Na,K-ATPase α1-subunit. Our results suggest that dexamethasone and insulin stimulate Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells was mediated by Na,K-ATPase synthesis and an increased enzymatic activity because of dephosphorylation of Na,K-ATPase α1-subunits. The effect of insulin is mediated by the protein kinase C, PP1, and/or PP2A pathways.

  6. Gremlin Activates the Smad Pathway Linked to Epithelial Mesenchymal Transdifferentiation in Cultured Tubular Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Raquel Rodrigues-Diez

    2014-01-01

    Full Text Available Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-β (TGF-β. Epithelial mesenchymal transition (EMT is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2 with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription. The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-β.

  7. ASSOCIATION OF CENTRAL CORNEAL THICKNESS AND CENTRAL CORNEAL ENDOTHELIAL CELL COUNT WITH PROGRESSIVE STAGES OF DIABETIC RETINOPATHY

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    Nirmal Kumar Sasmal

    2017-10-01

    Full Text Available BACKGROUND Diabetes mellitus along with its different serious pathological complications are increasing very rapidly in both developed and developing countries and manifesting as an escalating pandemic leading to morbidity and untimely death. This may lead to irreversible socioeconomic and psychosocial damage to the individuals, families and communities, either directly or indirectly. Assessment of Central Corneal Thickness (CCT and Central Corneal Endothelial Cell Count (CCEC in different stages of DR would help us to understand how rapidly the disease spreads, which could in turn be used as guidelines for disease screening in patients as risk to developing the disease. The aim of the study is to understand the association of CCT and CECC with progressive stages of Diabetic Retinopathy (DR in Diabetes Mellitus type 2 (DM-2. MATERIALS AND METHODS A case-control study was performed with a random eye from cases, No Diabetic Retinopathy (NDR, Nonproliferative Diabetic Retinopathy (NPDR and Proliferative Diabetic Retinopathy (PDR to determine association of DR with CCT and CECC. Parameters were quantified by ultrasonic pachymeter and specular microscope and results statistically analysed to understand significant association. RESULTS Significant increase in CCT was observed in NPDR and PDR compared to controls or NDR. Conversely, NPDR and PDR showed significant decrease compared to controls or NDR. CCT and CECC showed significant inverse correlation in all groups. CONCLUSION CCT and CECC showed significant increase and decrease respectively with stages of DR and were inversely correlated with each other. Assessment of CCT and CECC could thus be used as effective indicators of ocular manifestations of DM for early therapeutic intervention.

  8. Regulated Mucin Secretion from Airway Epithelial Cells

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    Kenneth Bruce Adler

    2013-09-01

    Full Text Available Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3x10^6 D per monomer whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ~1 um in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among MARCKS, cysteine string protein (CSP, HSP70 and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG. Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the

  9. Andrographolide suppresses epithelial mesenchymal transition by inhibition of MAPK signalling pathway in lens epithelial cells.

    Science.gov (United States)

    Kayastha, Forum; Johar, Kaid; Gajjar, Devarshi; Arora, Anshul; Madhu, Hardik; Ganatra, Darshini; Vasavada, Abhay

    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-beta 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 alpha-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.

  10. 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.

  11. Gill epithelial cells as in vitro models in aquatic toxicology.

    Science.gov (United States)

    Sandbacka, M; Christianson, I; Isomaa, B

    2000-01-01

    Gill epithelial cells are less sensitive than fish for most test chemicals, but a high correlation and a slope of the regression line close to 1 support the use of gill epithelial cells for prediction of acute toxicity in fish. Cells in suspension perform as well as cultured cells in the toxicity tests. However, the use of cells in suspension results in a quicker and more cost-effective assay for toxicity screening, but the cells should be used within about 5 hours of isolation. If a longer incubation time is required, cultured cells should be used. Cultured cells re-establish their polarity and contacts with other cells, and retain detectable amounts of enzymes for xenobiotic metabolism for at least 12 days in culture. Epithelial cell layers grown on filters seem to be less suitable for toxicity screening. 2000 FRAME.

  12. Cell volume regulation in epithelial physiology and cancer

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Hoffmann, Else Kay; Novak, Ivana

    2013-01-01

    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...... 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....

  13. 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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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. (paper)

  15. Resident corneal c-fms(+) macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis.

    Science.gov (United States)

    Ramke, Mirja; Zhou, Xiaohong; Materne, Emma Caroline; Rajaiya, Jaya; Chodosh, James

    2016-06-01

    The cornea contains a heterogeneous population of antigen-presenting cells with the capacity to contribute to immune responses. Adenovirus keratitis is a severe corneal infection with acute and chronic phases. The role of resident corneal antigen-presenting cells in adenovirus keratitis has not been studied. We utilized transgenic MaFIA mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, in a mouse model of adenovirus keratitis. Clinical keratitis and recruitment of myeloperoxidase and CD45(+) cells were diminished in c-fms depleted, adenovirus infected mice, as compared to controls, consistent with a role for myeloid-lineage cells in adenovirus keratitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Resident corneal c-fms+ macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis

    Science.gov (United States)

    Ramke, Mirja; Zhou, Xiaohong; Materne, Emma Caroline; Rajaiya, Jaya; Chodosh, James

    2016-01-01

    The cornea contains a heterogeneous population of antigen-presenting cells with the capacity to contribute to immune responses. Adenovirus keratitis is a severe corneal infection with acute and chronic phases. The role of resident corneal antigen-presenting cells in adenovirus keratitis has not been studied. We utilized transgenic MaFIA mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, in a mouse model of adenovirus keratitis. Clinical keratitis and recruitment of myeloperoxidase and CD45+ cells were diminished in c-fms depleted, adenovirus infected mice, as compared to controls, consistent with a role for myeloid-lineage cells in adenovirus keratitis. PMID:27185163

  17. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Activation of lung epithelial cells by group 2 mite allergens

    OpenAIRE

    Österlund, Camilla

    2012-01-01

    Throughout many parts of the world house dust mites (HDM) are considered as a major source of indoor aeroallergens and they are powerful inducers of allergic diseases. Proteolytic HDM allergens are recognised as being able to directly activate respiratory epithelial cells and thereby actively participate in innate immune responses. Although several major HDM allergens lack proteolytic activity, their possible ability to similarly interact with epithelial cells is not known. The overall aim of...

  19. Renal disease, epidermal necrosis, and epithelial cell antibodies.

    OpenAIRE

    Deal, J E; Groves, R W; Harmer, A W; Welsh, K I; MacDonald, D M; Rigden, S P

    1991-01-01

    OBJECTIVE--To describe the association between epithelial cell IgM, which has previously been associated with an increased incidence of loss of renal graft in children, with a novel cutaneous eruption and unexplained native renal disease. DESIGN--Observational study on children with epithelial cell antibody presenting with unexplained renal or skin disease. SETTING--General paediatric department and regional paediatric nephrology unit. PATIENTS--Six children (five girls, one boy), who present...

  20. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with 3 H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-β did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  1. 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.

  2. Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

    Science.gov (United States)

    Maggio, Savannah; Takeda, Kazuyo; Stark, Felicity; Meierovics, Anda I.; Yabe, Idalia; Cowley, Siobhan C.

    2015-01-01

    The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates. PMID:26379269

  3. Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Savannah Maggio

    Full Text Available The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells. Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates.

  4. Rat glomerular epithelial cells in culture. Parietal or visceral epithelial origin

    International Nuclear Information System (INIS)

    Norgaard, J.O.

    1987-01-01

    Isolated glomeruli from rats were explanted under standard culture conditions and outgrowths were studied by light and electron microscopy in order to identify the cells. Rat glomerular samples contained 20 to 30% structurally well-preserved encapsulated glomeruli which had a large rate of attachment to the substrate and very constantly gave rise to cellular outgrowth. In order to label cells from which outgrowth originated the glomerular incorporation of [ 3 H]thymidine was studied in the preattachment phase. By light and electron microscope autoradiograph it was demonstrated that label was located only over visceral and parietal epithelial cells during the first 3 days of culture. Incorporation of [ 3 H]thymidine was seen in mesangial cells after 5 days, i.e., after the glomeruli had attached to the culture vessels and the initial outgrowth had appeared. Consequently the first cells to grow out were of epithelial origin. Glomeruli were then incubated with [ 3 H]thymidine for the first 2 1/2 days of culture in order to label the epithelial cells, then were allowed to attach to the substrate and induce cell outgrowth. By light microscope autoradiography performed with the outgrowths in situ two types of cells with labeled nuclei were seen: (a) a small, polyhedral ciliated cell which grew in colonies where the cells were joined by junctional complexes (type I), and (b) a second very large, often multinucleated cell (type II). Based on the structural resemblance with their counterparts in situ and on comparisons with positively identified visceral epithelial cells in outgrowths from other species it is suggested that type I cells are derived from the parietal epithelium of Bowman's capsule and type II cells from the visceral epithelium

  5. 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

  6. 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

  7. From cells to tissue: A continuum model of epithelial mechanics

    Science.gov (United States)

    Ishihara, Shuji; Marcq, Philippe; Sugimura, Kaoru

    2017-08-01

    A two-dimensional continuum model of epithelial tissue mechanics was formulated using cellular-level mechanical ingredients and cell morphogenetic processes, including cellular shape changes and cellular rearrangements. This model incorporates stress and deformation tensors, which can be compared with experimental data. Focusing on the interplay between cell shape changes and cell rearrangements, we elucidated dynamical behavior underlying passive relaxation, active contraction-elongation, and tissue shear flow, including a mechanism for contraction-elongation, whereby tissue flows perpendicularly to the axis of cell elongation. This study provides an integrated scheme for the understanding of the orchestration of morphogenetic processes in individual cells to achieve epithelial tissue morphogenesis.

  8. 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.

  9. Cells from the adult corneal stroma can be reprogrammed to a neuron-like cell using exogenous growth factors

    International Nuclear Information System (INIS)

    Greene, Carol Ann; Chang, Chuan-Yuan; Fraser, Cameron J.; Nelidova, Dasha E.; Chen, Jing A.; Lim, Angela; Brebner, Alex; McGhee, Jennifer; Sherwin, Trevor; Green, Colin R.

    2014-01-01

    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

  10. 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.

  11. Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology

    Energy Technology Data Exchange (ETDEWEB)

    Lasalvia, Maria [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Castellani, Stefano [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy); D’Antonio, Palma [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Perna, Giuseppe [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Carbone, Annalucia [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy); Colia, Anna Laura; Maffione, Angela Bruna [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Capozzi, Vito [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Conese, Massimo, E-mail: massimo.conese@unifg.it [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy)

    2016-10-15

    The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. - Highlights: • CF bronchial epithelial (CFBE) cells show a disorganized actin cytoskeleton. • CFBE cells present high roughness and low rigidity in

  12. Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology

    International Nuclear Information System (INIS)

    Lasalvia, Maria; Castellani, Stefano; D’Antonio, Palma; Perna, Giuseppe; Carbone, Annalucia; Colia, Anna Laura; Maffione, Angela Bruna; Capozzi, Vito; Conese, Massimo

    2016-01-01

    The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. - Highlights: • CF bronchial epithelial (CFBE) cells show a disorganized actin cytoskeleton. • CFBE cells present high roughness and low rigidity in

  13. A Dual Role for Corneal Dendritic Cells in Herpes Simplex Keratitis: Local Suppression of Corneal Damage and Promotion of Systemic Viral Dissemination

    Science.gov (United States)

    Hu, Kai; Harris, Deshea L.; Yamaguchi, Takefumi; von Andrian, Ulrich H.; Hamrah, Pedram

    2015-01-01

    The cornea is the shield to the foreign world and thus, a primary site for peripheral infections. However, transparency and vision are incompatible with inflammation and scarring that may result from infections. Thus, the cornea is required to perform a delicate balance between fighting infections and preserving vision. To date, little is known about the specific role of antigen-presenting cells in viral keratitis. In this study, utilizing an established murine model of primary acute herpes simplex virus (HSV)-1 keratitis, we demonstrate that primary HSV keratitis results in increased conventional dendritic cells (cDCs) and macrophages within 24 hours after infection. Local depletion of cDCs in CD11c-DTR mice by subconjuntival diphtheria toxin injections, led to increased viral proliferation, and influx of inflammatory cells, resulting in increased scarring and clinical keratitis. In addition, while HSV infection resulted in significant corneal nerve destruction, local depletion of cDCs resulted in a much more severe loss of corneal nerves. Further, local cDC depletion resulted in decreased corneal nerve infection, and subsequently decreased and delayed systemic viral transmission in the trigeminal ganglion and draining lymph node, resulting in decreased mortality of mice. In contrast, sham depletion or depletion of macrophages through local injection of clodronate liposomes had neither a significant impact on the cornea, nor an effect on systemic viral transmission. In conclusion, we demonstrate that corneal cDCs may play a primary role in local corneal defense during viral keratitis and preserve vision, at the cost of inducing systemic viral dissemination, leading to increased mortality. PMID:26332302

  14. 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.

  15. 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.

  16. Human renal tubular epithelial cells suppress alloreactive T cell proliferation.

    Science.gov (United States)

    Demmers, M W H J; Korevaar, S S; Roemeling-van Rhijn, M; van den Bosch, T P P; Hoogduijn, M J; Betjes, M G H; Weimar, W; Baan, C C; Rowshani, A T

    2015-03-01

    Renal tubular epithelial cells (TECs) are one of the main targets of alloreactive T cells during acute rejection. We hypothesize that TECs modulate the outcome of alloimmunity by executing immunosuppressive effects in order to dampen the local inflammation. We studied whether TECs possess immunosuppressive capacities and if indoleamine 2,3-dioxygenase (IDO) might play a role in suppressing T cell alloreactivity. Next, we studied the role of programmed death ligand 1 (PD-L1) and intercellular adhesion molecule-1 (ICAM-1 with regard to TEC-related immunomodulatory effects. CD3/CD28 and alloactivated peripheral blood mononuclear cells were co-cultured with activated TECs. We analysed CD4(+) and CD8(+) T cell proliferation and apoptosis in the absence or presence of IDO inhibitor 1-methyl-L-tryptophan (1-L-MT), anti-PD-L1 and anti-ICAM-1. Further, we examined whether inhibition of T cell proliferation was cell-cell contact-dependent. We found that TECs dose-dependently inhibited CD4(+) and CD8(+) T cell proliferation (Pcell proliferation was only partially restored or failed to restore using 1-L-MT. Activated TECs increased early and late apoptosis of proliferating CD4(+) and CD8(+) T cells; only CD4(+) T cell apoptosis was statistically affected by 1-L-MT. Transwell experiments revealed that TEC-mediated immunosuppression is cell-cell contact-dependent. We found that anti-ICAM-1 affected only CD4(+) T cell apoptosis and not T cell proliferation. Our data show that TECs suppress both CD4(+) and CD8(+) T cell proliferation contact dependently. Interestingly, inhibition of proliferation and enhancement of apoptosis of T cell subsets is differentially regulated by indoleamine 2,3-dioxygenase and ICAM-1, with no evidence for the involvement of PD-L1 in our system. © 2014 British Society for Immunology.

  17. Indoleamine 2,3-dioxygenase protects corneal endothelial cells from UV mediated damage.

    Science.gov (United States)

    Serbecic, Nermin; Beutelspacher, Sven Christoph

    2006-03-01

    Indoleamine-2,3-dioxygenase (IDO) is an intracellular enzyme present in dendritic cells and macrophages. It is a known modulator of T-cell response and contributes to the UV protection of the lens. There yet is no information on IDO activity in the corneal endothelium, protecting the endothelial cells from light mediated damage. We exposed murine corneal endothelial cells (MCEC) with different doses of UV-B light 280-320 nm, probed for IDO mRNA (real-time PCR) and assessed apoptosis rate (flow cytometry) and caspase-3-activity in the cells. The metabolites of the IDO catalysed reaction, l-kynurenine, was also measured. Malondialdehyde was detected for quantification of UV-B-induced oxidative stress. To investigate specificity, IDO effects were blocked by 1-methyl-tryptophan. The effects of IDO overexpression in the MCEC were assessed by transfection of an expression vector. MCEC consistently express IDO at low levels. Exposure to UV-B light led to a dose-responding upregulation of IDO; IDO was found competent converting l-tryptophan into l-kynurenine. Irradiation led to increased apoptosis and caspase-3-activity of MCEC. Supplementation of l-kynurenine or overexpression of IDO in the MCEC could reduce apoptosis significantly following UV-B irradiation. Inhibition of IDO by 1-MT was potent to reverse this effect. IDO and its metabolite l-kynurenine can protect corneal endothelial cells from UV-B-induced oxidative stress and apoptosis. It may be an active protection mechanism against corneal endothelial damage.

  18. Human lung epithelial cells A549 epithelial-mesenchymal transition induced by PVA/Collagen nanofiber.

    Science.gov (United States)

    Li, Xiuchun; Yan, Shanshan; Dai, Jing; Lu, Yi; Wang, Yiqun; Sun, Man; Gong, Jinkang; Yao, Yuan

    2018-02-01

    Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell-cell contact to become mesenchymal stem cells, which is important on development and embryogenesis, wound healing, and cancer metastasis. This research aims to investigate the effect of topological cue as modulating factor on the EMT by tuning the diameter of electrospinning nanofiber. The cell-nanofiber interaction between human lung epithelial cell A549 and electrospinning nanofibers composed of polyvinyl alcohol (PVA) and type I collagen were investigated. The electrospinning of regenerated PVA/Collagen nanofibers were performed with water/acetic acid as a spinning solvent and glutaraldehyde as a chemical cross-linker. Parameterization on concentration, applied voltage and feeding rate was finalized to generate smooth nanofibers with good homogeneity. The scanning electron microscopy result demonstrated that A549 cell appropriately achieved extended morphology by the filopodia attaching to the surface of the nanofibrous mats. When the diameter changed from 90nm to 240nm, the A549 cell was correspondingly express varied EMT related genes. Gene expression analysis was conducted by qPCR using three typical markers for detecting EMT: N-cadherin (NCad), Vimentin (Vim), and Fibronectin (Fib). An increasing expression pattern was observed on cell culturing on 170nm sample with respect to cell cultured on 90nm and 240nm. This result indicated the 170nm PVA/Collagen nanofibers induce A549 cells to process epithelial-mesenchymal transition more seriously than those on 90nm or 240nm. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Toxic mechanisms of copper oxide nanoparticles in epithelial kidney cells

    DEFF Research Database (Denmark)

    Thit, Amalie; Selck, Henriette; Bjerregaard, Henning F.

    2015-01-01

    CuO NPs have previously been reported as toxic to a range of cell cultures including kidney epithelial cells from the frog, Xenopus laevis (A6). Here we examine the molecular mechanisms affecting toxicity of Cu in different forms and particle sizes. A6 cells were exposed to ionic Cu (Cu2+) or Cu...

  20. Cholera toxin stimulation of human mammary epithelial cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  1. The regenerative potential of parietal epithelial cells in adult mice

    NARCIS (Netherlands)

    Berger, K.; Schulte, K.; Boor, P.; Kuppe, C.; Kuppevelt, T.H. van; Floege, J.; Smeets, B.; Moeller, M.J.

    2014-01-01

    Previously, we showed that some podocytes in juvenile mice are recruited from cells lining Bowman's capsule, suggesting that parietal epithelial cells (PECs) are a progenitor cell population for podocytes. To investigate whether PECs also replenish podocytes in adult mice, PECs were genetically

  2. Extracellular cleavage of E-cadherin promotes epithelial cell extrusion

    NARCIS (Netherlands)

    Grieve, Adam G; Rabouille, Catherine

    2014-01-01

    Epithelial cell extrusion and subsequent apoptosis is a key mechanism to prevent the accumulation of excess cells. By contrast, when driven by oncogene expression, apical cell extrusion is followed by proliferation and represents an initial step of tumorigenesis. E-cadherin (E-cad), the main

  3. CD40 is functionally expressed on human thymic epithelial cells

    NARCIS (Netherlands)

    Galy, A. H.; Spits, H.

    1992-01-01

    CD40 is a prominent B cell Ag also found on certain epithelial cells and on carcinomas. In this report, we analyzed CD40 distribution in the human thymus. CD40 was not found on the majority of CD45-positive thymocytes, but was present in a CD45-negative stromal cell population. Immunohistology

  4. Personalized Stem Cell Therapy to Correct Corneal Defects Due to a Unique Homozygous-Heterozygous Mosaicism of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

    Science.gov (United States)

    Barbaro, Vanessa; Nasti, Annamaria Assunta; Raffa, Paolo; Migliorati, Angelo; Nespeca, Patrizia; Ferrari, Stefano; Palumbo, Elisa; Bertolin, Marina; Breda, Claudia; Miceli, Francesco; Russo, Antonella; Caenazzo, Luciana; Ponzin, Diego; Palù, Giorgio; Parolin, Cristina; Di Iorio, Enzo

    2016-08-01

    : Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches. This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative

  5. Metabolic re-wiring of isogenic breast epithelial cell lines following epithelial to mesenchymal transition.

    Science.gov (United States)

    Halldorsson, Skarphedinn; Rohatgi, Neha; Magnusdottir, Manuela; Choudhary, Kumari Sonal; Gudjonsson, Thorarinn; Knutsen, Erik; Barkovskaya, Anna; Hilmarsdottir, Bylgja; Perander, Maria; Mælandsmo, Gunhild M; Gudmundsson, Steinn; Rolfsson, Óttar

    2017-06-28

    Epithelial to mesenchymal transition (EMT) has implications in tumor progression and metastasis. Metabolic alterations have been described in cancer development but studies focused on the metabolic re-wiring that takes place during EMT are still limited. We performed metabolomics profiling of a breast epithelial cell line and its EMT derived mesenchymal phenotype to create genome-scale metabolic models descriptive of both cell lines. Glycolysis and OXPHOS were higher in the epithelial phenotype while amino acid anaplerosis and fatty acid oxidation fueled the mesenchymal phenotype. Through comparative bioinformatics analysis, PPAR-γ1, PPAR- γ2 and AP-1 were found to be the most influential transcription factors associated with metabolic re-wiring. In silico gene essentiality analysis predicts that the LAT1 neutral amino acid transporter is essential for mesenchymal cell survival. Our results define metabolic traits that distinguish an EMT derived mesenchymal cell line from its epithelial progenitor and may have implications in cancer progression and metastasis. Furthermore, the tools presented here can aid in identifying critical metabolic nodes that may serve as therapeutic targets aiming to prevent EMT and inhibit metastatic dissemination. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  6. 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.

  7. Melatonin modulates microfilament phenotypes in epithelial cells, implications for adhesion and inhibition of cancer cell migration

    OpenAIRE

    Benítez-King, Gloria; Soto-Vega, Elena; Ramírez-Rodriguez, Gerardo

    2009-01-01

    Cell migration and adhesion are cytoskeleton- dependent functions that play a key role in epithelial physiology. Specialized epithelial cells in water transport have specific microfilament rearrangements that make these cells adopt a polyhedral shape, forming a sealed monolayer which functions as permeability barrier. Also, specific polarized microfilament phenotypes are formed at the front and the rear of migratory epithelial cells. In pathological processes such a...

  8. Ultraviolet induced lysosome activity in corneal epithelium

    International Nuclear Information System (INIS)

    Cullen, A.P.

    1980-01-01

    A 5.000 W Xe-Hg high pressure lamp and a double monochromator were used to produce a 3.3 nm half-bandpass ultraviolet radiation at 295 nm. Pigmented rabbit eyes were irradiated with radiant exposures from 140 Jm -2 to 10.000 Jm -2 and evaluated by slit-lamp biomicroscopy, light and electron microscopy. Corneal threshold (Hsub(c) was 200 Jm -2 and lens threshold (Hsub(L)) was 7.500 Jm -2 . The most repeatable and reliable corneal response to these levels of UV was the development of corneal epithelial granules. Histological changes included a loss of superficial epithelial cells and selective UV induced autolysis of the wing cells. It is suggested that the biomicroscopically observed granules are the clinical manifestation of the secondary lysosomes revealed by light and electron microscopy. It is proposed that UV breaks down the primary lysosome membranes to release hydrolytic enzymes which in turn form the secondary lysosomes during autolysis. Extreme levels of radiant exposure at 295 nm result in indiscriminate destruction of all layers of the corneal epithelium, but the posterior cornea was spared. (orig.) [de

  9. 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.

  10. Fungal glycan interactions with epithelial cells in allergic airway disease.

    Science.gov (United States)

    Roy, René M; Klein, Bruce S

    2013-08-01

    Human exposure to fungi results in a wide range of health outcomes, from invasive disease or allergy to immune tolerance. Inhaled fungi contact airway epithelial cells as an early event, and this host:fungal interaction can shape the eventual immunological outcome. Emerging evidence points to exposure to fungal cell wall carbohydrates in the development of allergic airway disease. Herein, we describe determinants of fungal allergenicity, and review the responses of airway epithelial cells to fungal carbohydrates. A greater understanding of the recognition of and response to fungal carbohydrates by airway epithelial cells may lead to the development of targeted therapies that ameliorate allergic airway disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. 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

    Diffusion gradients of morphogens have been inferred as a basis for the control of morphogenesis in hydra, and morphogenetic substances have been found which, on the basis of their molecular weight (MW), should be able to pass gap junctions. There have been several reports of the presence of gap...... 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...

  12. Urea selectively induces DNA synthesis in renal epithelial cells.

    Science.gov (United States)

    Cohen, D M; Gullans, S R

    1993-04-01

    Hyperosmotic stress with the functionally impermeant solute NaCl has been shown by us and others to inhibit cell growth and DNA synthesis. Several lines of evidence suggest that urea, the other principal renal medullary solute, may exert a growth-promoting effect on renal epithelial cells. Among these is the finding that urea upregulates expression at the mRNA level of two growth-associated immediate-early genes, Egr-1 and c-fos. In the present study, urea, in concentrations characteristic of the renal medulla, increased [3H]thymidine incorporation approximately threefold in confluent, growth-suppressed Madin-Darby canine kidney (MDCK) cells, whereas another readily membrane-permeant solute, glycerol, did not. Urea also overcame the inhibitory effect of hyperosmotic NaCl on DNA synthesis. The urea-induced increase in [3H]thymidine incorporation was also evident in the renal epithelial LLC-PK1 cell line, but not in renal nonepithelial and epithelial nonrenal cell types examined. In addition, it was associated with a 15% increase in total DNA content measured fluorometrically at 24 h of treatment. There was, however, no associated increase in cell proliferation as measured by cell number, total protein content, or cell cycle distribution. Urea also failed to induce polyploidy or aneuploidy. Therefore cells of renal epithelial origin may be uniquely capable of responding to hyperosmotic urea with increased DNA synthesis through an undefined and potentially novel mechanism.

  13. 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...

  14. Comparison of the corneal endothelial cell count in type II diabetic patients with healthy adults

    International Nuclear Information System (INIS)

    Rizvi, B.Z.; Zafar, O.

    2016-01-01

    To compare the mean corneal endothelial cell count in type II diabetic patients with healthy adults. Study Design: Case control. Place and Duration of Study: Out-patient Department of Armed Forces Institute of Ophthalmology, Rawalpindi from September 10, 2013 to March 25, 2014. Material and Methods: A hospital-based case-control study was carried out at out-patient department of Armed Forces Institute of Ophthalmology in which 130 eyes (65 diabetic eyes and 65 controls) were included. Non-probability consecutive sampling was adopted. Relevant detailed history including information about age, gender, duration of diabetes, any other medical illness and current medical treatment being taken by patient was recorded. Results: Data entry and analysis was done in SPSS version 10. Total 130 eyes (65 diabetic and 65 non-diabetic eyes) were included in our study according to the inclusion criteria. Mean age (years) of patient in both the groups was 59.55 +- 8.01 and 53.85 +- 10.07. Mean corneal endothelial cell count in both the groups was 2368.35 +- 389.58 and 2588.64 +- 269.84 respectively which was statistically significant (p-value=0.001) in both the groups. Conclusion: The conclusion of the study was that the mean corneal endothelial cell count in type II diabetic patients was significantly less as compared to healthy adults. (author)

  15. Corneal Ulcer

    Science.gov (United States)

    ... Stories Español Eye Health / Eye Health A-Z Corneal Ulcer Sections What Is a Corneal Ulcer? Corneal Ulcer ... Ulcer Diagnosis Corneal Ulcer Treatment What Is a Corneal Ulcer? Leer en Español: ¿Qué es una úlcera de ...

  16. Formation of reactive oxygen species in rat epithelial cells upon ...

    Indian Academy of Sciences (India)

    Unknown

    < 100 nm) that contributed 31% to the particle number. In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a.

  17. Formation of reactive oxygen species in rat epithelial cells upon ...

    Indian Academy of Sciences (India)

    In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a clear dose-response relationship concerning the ...

  18. Vulnerability of Normal Human Mammary Epithelial Cells to Oncogenic Transformation

    Science.gov (United States)

    2012-04-01

    cells. Proc Nat Acad Sci USA 108:3264-69. 2011 Chin, K, Ortiz de Solorzano , C, Knowles, D, Jones, A, Chou, W, Rodriguez, E, Kuo, W-L, Ljung, B-M...Transformation of human mammary epithelial cells by oncogenic retro- viruses. Cancer Res 1988;48:4689–94. 13. Chin K, de Solorzano CO, Knowles D, et al

  19. A murine and a porcine coronavirus are released from opposite surfaces of the same epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Bekker, C P; Strous, G J; Horzinek, M C; Dveksler, G S; Holmes, K V; Rottier, P J

    1996-01-01

    Epithelial cells are important target cells for coronavirus infection. Earlier we have shown that transmissible gastroenteritis coronavirus (TGEV) and mouse hepatitis coronavirus (MHV) are released from different sides of porcine and murine epithelial cells, respectively. To study the release of

  20. Ghrelin inhibits ovarian epithelial carcinoma cell proliferation in vitro.

    Science.gov (United States)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang; Zhang, Yi

    2013-11-01

    The only orexigenic peptide, ghrelin, which is primarily produced by the gastrointestinal tract, has been implicated in malignant cell proliferation and invasion. Ghrelin is a natural ligand of the growth hormone secretagogue receptor 1a (GHSR1a). However, the role of ghrelin in ovarian epithelial carcinoma remains unknown since the expression of GHSR1a in ovary is not confirmed. The aim of the present study was to assess expression of ghrelin and its receptor in human ovarian epithelial carcinoma and to examine the effect of ghrelin on carcinoma cell proliferation. Frozen sections of ovarian samples and the human ovarian epithelial carcinoma cell line, HO-8910, were used to characterize the expression of ghrelin/GHSR1a axis and the effect of ghrelin on proliferation. We found that ghrelin and GHSR1a are expressed in ovarian epithelial carcinoma in vivo and in vitro. Ghrelin inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, and this inhibition may be abolished by the ghrelin receptor antagonist D-Lys-3-GH-releasing peptide-6 and ghrelin neutralizing antibody. Ghrelin enhances HO-8910 cell apoptosis and autophagy. The activation of mammalian target of rapamycin (mTOR) signaling pathway blocks the effects of ghrelin-induced autophagy and apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation induced by ghrelin. In conclusion, the present study demonstrates that ghrelin inhibits the proliferation of human HO-8910 ovarian epithelial carcinoma cells by inducing apoptosis and autophagy via the mTOR signaling pathway. This study provides a novel regulatory signaling pathway of ghrelin-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy.

  1. Alterations in the corneal nerve and stem/progenitor cells in diabetes: preventive effects of insulin-like growth factor-1 treatment.

    Science.gov (United States)

    Ueno, Hiroki; Hattori, Takaaki; Kumagai, Yuta; Suzuki, Noboru; Ueno, Satoki; Takagi, Hitoshi

    2014-01-01

    This study aimed to investigate whether corneal nerve and corneal stem/progenitor cells are altered in insulin-like growth factor-I (IGF-I-) treated individuals with diabetes. A group consisting of db/db mice with type 2 diabetes mellitus (DM) and a wild-type group were assessed by neural and corneal stem/progenitor cell markers immunostaining and real-time PCR. Moreover, the expression of corneal nerve and stem/progenitor cell markers was examined in IGF-1-treated diabetic mice. Compared with a normal cornea, swelling and stratification of the corneal epithelium were noted in db/db mice. Beta-III tubulin immunostaining revealed that the corneal subbasal plexuses in diabetic mice were thinner with fewer branches. mRNA expression levels of Hes1, Keratin15, and p75 (corneal stem/progenitor cell markers) and the intensity and number of positive cells of Hes1 and Keratin19 immunostaining diminished in the diabetic corneas. Compared with the subbasal nerve density in the normal group, a decrease in the diabetic group was observed, whereas the corneal subbasal nerve density increased in IGF-1-treated diabetic group. The decreased expression of Hes1 and Keratin19 was prevented in IGF-1-treated diabetic group. Our data suggest that corneal nerve and stem/progenitor cells are altered in type 2 DM, and IGF-I treatment is capable of protecting against corneal damage in diabetes.

  2. Alterations in the Corneal Nerve and Stem/Progenitor Cells in Diabetes: Preventive Effects of Insulin-Like Growth Factor-1 Treatment

    Directory of Open Access Journals (Sweden)

    Hiroki Ueno

    2014-01-01

    Full Text Available This study aimed to investigate whether corneal nerve and corneal stem/progenitor cells are altered in insulin-like growth factor-I (IGF-I- treated individuals with diabetes. A group consisting of db/db mice with type 2 diabetes mellitus (DM and a wild-type group were assessed by neural and corneal stem/progenitor cell markers immunostaining and real-time PCR. Moreover, the expression of corneal nerve and stem/progenitor cell markers was examined in IGF-1-treated diabetic mice. Compared with a normal cornea, swelling and stratification of the corneal epithelium were noted in db/db mice. Beta-III tubulin immunostaining revealed that the corneal subbasal plexuses in diabetic mice were thinner with fewer branches. mRNA expression levels of Hes1, Keratin15, and p75 (corneal stem/progenitor cell markers and the intensity and number of positive cells of Hes1 and Keratin19 immunostaining diminished in the diabetic corneas. Compared with the subbasal nerve density in the normal group, a decrease in the diabetic group was observed, whereas the corneal subbasal nerve density increased in IGF-1-treated diabetic group. The decreased expression of Hes1 and Keratin19 was prevented in IGF-1-treated diabetic group. Our data suggest that corneal nerve and stem/progenitor cells are altered in type 2 DM, and IGF-I treatment is capable of protecting against corneal damage in diabetes.

  3. Serum-Free Cryopreservation of Human Amniotic Epithelial Cells

    OpenAIRE

    H. Niknejad; H. Peirovi; B. Jambar Noushin

    2013-01-01

    Introduction & Objective: One of the important issues in long term storage of cells is removal of animal serum from cell culture environments. The aim of this study was to evaluate amni-otic fluid (AF), which is full of growth factors, as substitute for fetal bovine serum (FBS) in the cryopreservation protocol. Materials & Methods: In this experimental study human amniotic epithelial cells were isolated from placentas which were seronegative for microbial infections. The cells were preserved ...

  4. 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

  5. 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

  6. 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.

  7. Intestinal Epithelial Cells Synthesize Glucocorticoids and Regulate T Cell Activation

    Science.gov (United States)

    Cima, Igor; Corazza, Nadia; Dick, Bernhard; Fuhrer, Andrea; Herren, Simon; Jakob, Sabine; Ayuni, Erick; Mueller, Christoph; Brunner, Thomas

    2004-01-01

    Glucocorticoids (GCs) are important steroid hormones with widespread activities in metabolism, development, and immune regulation. The adrenal glands are the major source of GCs and release these hormones in response to psychological and immunological stress. However, there is increasing evidence that GCs may also be synthesized by nonadrenal tissues. Here, we report that the intestinal mucosa expresses steroidogenic enzymes and releases the GC corticosterone in response to T cell activation. T cell activation causes an increase in the intestinal expression of the steroidogenic enzymes required for GC synthesis. In situ hybridization analysis revealed that these enzymes are confined to the crypt region of the intestinal epithelial layer. Surprisingly, in situ–produced GCs exhibit both an inhibitory and a costimulatory role on intestinal T cell activation. In the absence of intestinal GCs in vivo, activation by anti-CD3 injection resulted in reduced CD69 expression and interferon-γ production by intestinal T cells, whereas activation by viral infection led to increased T cell activation. We conclude that the intestinal mucosa is a potent source of immunoregulatory GCs. PMID:15596520

  8. Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Anja K Gruenert

    Full Text Available Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2 vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss- DNA vector genome into double-stranded (ds- DNA. This step can be bypassed by using self-complementary (sc- AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV and 38.0±8.6% (ssAAV (p<0.001, respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic.

  9. 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

    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

  10. Proliferating cells in HIV and pamidronate-associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells.

    NARCIS (Netherlands)

    Dijkman, H.B.P.M.; Weening, J.J.; Smeets, B.; Verrijp, K.; Kuppevelt, A.H.M.S.M. van; Assmann, K.K.; Steenbergen, E.; Wetzels, J.F.M.

    2006-01-01

    Collapsing focal segmental glomerulosclerosis (cFSGS) is characterized by hyperplasia of glomerular epithelial cells. In a mouse model of FSGS and in a patient with recurrent idiopathic FSGS, we identified the proliferating cells as parietal epithelial cells (PECs). In the present study, we have

  11. Proliferating cells in HIV and pamidronate-associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells

    NARCIS (Netherlands)

    Dijkman, H. B. P. M.; Weening, J. J.; Smeets, B.; Verrijp, K. C. N.; van Kuppevelt, T. H.; Assmann, K. K. J. M.; Steenbergen, E. J.; Wetzels, J. F. M.

    2006-01-01

    Collapsing focal segmental glomerulosclerosis (cFSGS) is characterized by hyperplasia of glomerular epithelial cells. In a mouse model of FSGS and in a patient with recurrent idiopathic FSGS, we identified the proliferating cells as parietal epithelial cells (PECs). In the present study, we have

  12. Corneal endothelial cell density and morphology in low and moderate myopic Chinese eyes

    Directory of Open Access Journals (Sweden)

    Jane Mei Chun

    2013-08-01

    Full Text Available AIM: To describe and compare the corneal endothelial cell density and morphology in young, low and moderate myopic Chinese adults in Malaysian Chinese population.METHODS: Non-contact specular microscopy (Topcon SP3000P, Tokyo, Japan was performed in low (n=78; 21.22±1.51 years and moderate (n=78; 21.82±1.40 years myopic subjects. The mean of three consecutive measurements of endothelial cell density (MCD, coefficient of variation (CV in the cell size, and hexagonal appearance of the cell were obtained.RESULTS: In low myopic eyes the MCD was 3 063.0±176.2/mm2, the mean CV was 33.4±4.0% and the mean hexagonal appearance of the cell was 57.9±2.7%. In moderate myopic eyes the MCD was 2961.6±159.0/mm2, the mean CV was 33.9±3.6% and mean hexagonal appearance of the cell was 56.2±4.7%. There were statistically significant differences in MCD (PPCONCLUSION:The corneal endothelial cell layer in more myopic eyes tends to have less MCD and cell hexagonality compared to lower myopic eyes. Nevertheless, there is no significant difference in CV between low and moderate myopic eyes.

  13. Apical trafficking in epithelial cells: signals, clusters and motors.

    Science.gov (United States)

    Weisz, Ora A; Rodriguez-Boulan, Enrique

    2009-12-01

    In the early days of epithelial cell biology, researchers working with kidney and/or intestinal epithelial cell lines and with hepatocytes described the biosynthetic and recycling routes followed by apical and basolateral plasma membrane (PM) proteins. They identified the trans-Golgi network and recycling endosomes as the compartments that carried out apical-basolateral sorting. They described complex apical sorting signals that promoted association with lipid rafts, and simpler basolateral sorting signals resembling clathrin-coated-pit endocytic motifs. They also noticed that different epithelial cell types routed their apical PM proteins very differently, using either a vectorial (direct) route or a transcytotic (indirect) route. Although these original observations have generally held up, recent studies have revealed interesting complexities in the routes taken by apically destined proteins and have extended our understanding of the machinery required to sustain these elaborate sorting pathways. Here, we critically review the current status of apical trafficking mechanisms and discuss a model in which clustering is required to recruit apical trafficking machineries. Uncovering the mechanisms responsible for polarized trafficking and their epithelial-specific variations will help understand how epithelial functional diversity is generated and the pathogenesis of many human diseases.

  14. Reversal of second-hand cigarette smoke-induced impairment of corneal wound healing by thymosin beta4 combined with anti-inflammatory agents.

    Science.gov (United States)

    Yuan, Hongwei; Ma, Chongze; Moinet, Lisa; Sato, Noboru; Martins-Green, Manuela

    2010-05-01

    Abnormalities in corneal reepithelialization caused by second-hand cigarette smoke (CS) are less known than the effects of CS on other tissues. The effects of CS on corneal epithelial cell migration and associated signaling mechanisms were examined, to determine the mechanisms by which CS delays corneal wound healing. Corneal epithelial cells in two-dimensional or organ culture were exposed to sidestream whole (SSW) smoke, a major component of second-hand CS. Thymosin beta 4 (Tbeta4), a molecule thought to promote wound healing in the cornea, was tested to determine whether it can reverse the adverse effects of SSW smoke on corneal healing. Cell migration, actin reorganization, and phosphorylation of focal adhesion kinase (FAK) and paxillin were all inhibited by exposure to SSW smoke, and the distribution of phospho-src in the cells was disrupted. Activation of RhoA, an important regulator of the cytoskeleton during cell migration, was also inhibited. Tbeta4 stimulated corneal epithelial cell migration in the presence of SSW smoke in culture and in vivo, and it partially reversed the inhibition of corneal healing by SSW smoke. However, Tbeta4 plus dexamethasone, an inhibitor of inflammation, together, reversed the effects of SSW smoke on corneal healing. These findings suggest that SSW smoke exerts its effects on cell migration during corneal epithelial healing through inhibition of actin reorganization, activation of focal adhesion molecules, formation of the focal adhesion complex, and activation of Rho-GTPases. Furthermore, they strongly suggest that corneal injury induced by toxicants can be treated using anti-inflammatory agents coupled with Tbeta4.

  15. Nicotine transport in lung and non-lung epithelial cells.

    Science.gov (United States)

    Takano, Mikihisa; Kamei, Hidetaka; Nagahiro, Machi; Kawami, Masashi; Yumoto, Ryoko

    2017-11-01

    Nicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Characteristics of [ 3 H]nicotine uptake was studied using these cell lines. Nicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations. The present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Oral epithelial cell reaction after exposure to Invisalign plastic material.

    Science.gov (United States)

    Premaraj, Thyagaseely; Simet, Samantha; Beatty, Mark; Premaraj, Sundaralingam

    2014-01-01

    Invisalign plastic aligners (Align Technology, Santa Clara, Calif) are used to correct malocclusions. The aligners wrap around the teeth and are in contact with gingival epithelium during treatment. The purpose of this study was to evaluate the cellular responses of oral epithelium exposed to Invisalign plastic in vitro. Oral epithelial cells were exposed to eluate obtained by soaking Invisalign plastic in either saline solution or artificial saliva for 2, 4, and 8 weeks. Cells grown in media containing saline solution or saliva served as controls. Morphologic changes were assessed by light microscopy. The 3-[4, 5-dimethythiazol- 2-yl]-2, 5-diphenyl tetrazolium bromide assay and flow cytometry were used to determine cell viability and membrane integrity, respectively. Cellular adhesion and micromotion of epithelial cells were measured in real time by electrical cell-substrate impedance sensing. Cells exposed to saline-solution eluate appeared rounded, were lifted from the culture plates, and demonstrated significantly increased metabolic inactivity or cell death (P <0.05). Saliva eluates did not induce significant changes in cell viability compared with untreated cells. Flow cytometry and electric cell-substrate impedance sensing showed that cells treated with saline-solution eluate exhibited compromised membrane integrity, and reduced cell-to-cell contact and mobility when compared with saliva-eluate treatment. Exposure to Invisalign plastic caused changes in viability, membrane permeability, and adhesion of epithelial cells in a saline-solution environment. Microleakage and hapten formation secondary to compromised epithelial integrity might lead to isocyanate allergy, which could be systemic or localized to gingiva. However, these results suggest that saliva might offer protection. Copyright © 2014 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

  17. Oxidative Stress, Cell Death, and Other Damage to Alveolar Epithelial Cells Induced by Cigarette Smoke

    Directory of Open Access Journals (Sweden)

    Nagai A

    2003-09-01

    Full Text Available Abstract Cigarette smoking is a major risk factor in the development of various lung diseases, including pulmonary emphysema, pulmonary fibrosis, and lung cancer. The mechanisms of these diseases include alterations in alveolar epithelial cells, which are essential in the maintenance of normal alveolar architecture and function. Following cigarette smoking, alterations in alveolar epithelial cells induce an increase in epithelial permeability, a decrease in surfactant production, the inappropriate production of inflammatory cytokines and growth factors, and an increased risk of lung cancer. However, the most deleterious effect of cigarette smoke on alveolar epithelial cells is cell death, i.e., either apoptosis or necrosis depending on the magnitude of cigarette smoke exposure. Cell death induced by cigarette smoke exposure can largely be accounted for by an enhancement in oxidative stress. In fact, cigarette smoke contains and generates many reactive oxygen species that damage alveolar epithelial cells. Whether apoptosis and/or necrosis in alveolar epithelial cells is enhanced in healthy cigarette smokers is presently unclear. However, recent evidence indicates that the apoptosis of alveolar epithelial cells and alveolar endothelial cells is involved in the pathogenesis of pulmonary emphysema, an important cigarette smoke-induced lung disease characterized by the loss of alveolar structures. This review will discuss oxidative stress, cell death, and other damage to alveolar epithelial cells induced by cigarette smoke.

  18. Prevotella intermedia ATCC 25611 targets host cell lamellipodia in epithelial cell adhesion and invasion.

    Science.gov (United States)

    Gursoy, U K; Könönen, E; Uitto, V-J

    2009-08-01

    The Prevotella intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, and Prevotella pallens, are phylogenetically closely related and potentially connected with oral and gastrointestinal tract disease pathogenesis. The aim of the present study was to examine whether these species differ in their capabilities of adhesion to and invasion of epithelial cells. Adhesion and invasion were assayed by standard antibiotic/culture assays and fluorescent microscopy techniques. The effect of Prevotella strains on epithelial cell viability was measured using a commercial cell proliferation assay. The strains P. intermedia ATCC 25611 and P. nigrescens ATCC 33263 adhered to epithelial cells, the adhesion numbers of P. intermedia being twice as high as those of P. nigrescens. These strains invaded epithelial cells but invasion was weak. The adhesion of P. intermedia was specifically targeted to epithelial cell lamellipodia. The number of adhered P. intermedia cells increased or decreased when the formation of lamellipodia was stimulated or inhibited, respectively. None of the tested strains showed toxic effects on epithelial cells; a clinical P. intermedia strain even increased the number of viable cells by about 20%. The results suggest that among the P. intermedia group bacteria, P. intermedia and P. nigrescens type strains can adhere to and invade epithelial cells, the capability of P. intermedia ATCC 25611(T) being highest in this context. This strain proved to have a special affinity in binding to epithelial cell lamellipodia.

  19. Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals.

    Science.gov (United States)

    Takezawa, Toshiaki; Nishikawa, Kazunori; Wang, Pi-Chao

    2011-09-01

    The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman's membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5-6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air-liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. 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

    2017-05-01

    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. Failure of most biomaterials originates from the inability to predict and control the influence of their surface properties on biological phenomena, particularly protein adsorption, and cellular behaviour, which subsequently results in unfavourable host response. Here, we introduce a surface-proteomic screening approach using a label-free mass spectrometry technique to decipher the adsorption profile of extracellular matrix (ECM) proteins on different biomaterials, and correlate it with cellular behaviour. We demonstrated that the way a biomaterial selectively interacts with specific ECM proteins of a given tissue seems to determine the interactions between the cells of that tissue and biomaterials. Accordingly, this approach can

  1. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    block alloactivation, had no inhibitory effect on RPE-mediated T-cell apoptotic responses in MHC class II-specific CD4+ T-cell lines. CONCLUSIONS: Retinal pigment epithelial cells express FasL and induce TCR-independent apoptosis in activated human T cells through Fas-FasL interaction. Retinal pigment...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  2. Airway epithelial cell response to human metapneumovirus infection

    International Nuclear Information System (INIS)

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-01-01

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-κB, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators

  3. The Regenerative Potential of Parietal Epithelial Cells in Adult Mice

    OpenAIRE

    Berger, Katja; Schulte, Kevin; Boor, Peter; Kuppe, Christoph; van Kuppevelt, Toin H.; Floege, Jürgen; Smeets, Bart; Moeller, Marcus J.

    2014-01-01

    Previously, we showed that some podocytes in juvenile mice are recruited from cells lining Bowman’s capsule, suggesting that parietal epithelial cells (PECs) are a progenitor cell population for podocytes. To investigate whether PECs also replenish podocytes in adult mice, PECs were genetically labeled in an irreversible fashion in 5-week-old mice. No significant increase in labeled podocytes was observed, even after 18 months. To accelerate a potential regenerative mechanism, progressive glo...

  4. Analysis of corneal endothelial cell density and morphology after laser in situ keratomileusis using two types of femtosecond lasers

    Directory of Open Access Journals (Sweden)

    Tomita M

    2012-09-01

    Full Text Available Minoru Tomita,1,2,* George O Waring IV,3,4 Miyuki Watabe,1,* 1Shinagawa LASIK Center, Chiyoda-ku, Tokyo, Japan; 2Department of Ophthalmology, Wenzhou Medical College, Wenzhou, China; 3Medical University of South Carolina, Storm Eye Institute, Charleston, SC, USA; 4Magill Laser Center, Charleston, SC, USA*These authors contributed equally to this studyPurpose: To compare two different femtosecond lasers used for flap creation during laser-assisted in situ keratomileusis (LASIK surgery in terms of their effects on the corneal endothelium.Methods: We performed LASIK surgery on 254 eyes of 131 patients using IntraLase FS60 (Abbott Medical Optics, Inc, Irvine, CA; IntraLase group and 254 eyes of 136 patients using Femto LDV (Ziemer Group AG, Port, Switzerland; LDV group for corneal flap creation. The mean cell density, coefficient of variation, and hexagonality of the corneal endothelial cells were determined and the results were statistically compared.Results: There were no statistically significant differences in the corneal morphology between pre and post LASIK results in each group, nor were there significant differences between the results of both groups at 3 months post LASIK.Conclusions: Both IntraLase FS60 and Ziemer Femto LDV are able to create flaps without significant adverse effects on the corneal endothelial morphology through 3 months after LASIK surgery.Keywords: LASIK, corneal endothelium, femtosecond laser, IntraLase FS60, Ziemer LDV

  5. Cytotoxic effects of air freshener biocides in lung epithelial cells.

    Science.gov (United States)

    Kwon, Jung-Taek; Lee, Mimi; Seo, Gun-Baek; Kim, Hyun-Mi; Shim, Ilseob; Lee, Doo-Hee; Kim, Taksoo; Seo, Jung Kwan; Kim, Pilje; Choi, Kyunghee

    2013-09-01

    This study evaluated the cytotoxicity of mixtures of citral (CTR) and either benzisothiazolinone (BIT, Mix-CTR-BIT) or triclosan (TCS, Mix-CTR-TCS) in human A549 lung epithelial cells. We investigated the effects of various mix ratios of these common air freshener ingredients on cell viability, cell proliferation, reactive oxygen species (ROS) generation, and DNA damage. Mix-CTR-BIT and Mix-CTR-TCS significantly decreased the viability of lung epithelial cells and inhibited cell growth in a dose-dependent manner. In addition, both mixtures increased ROS generation, compared to that observed in control cells. In particular, cell viability, growth, and morphology were affected upon increase in the proportion of BIT or TCS in the mixture. However, comet analysis showed that treatment of cells with Mix-CTR-BIT or Mix-CTR-TCS did not increase DNA damage. Taken together, these data suggested that increasing the content of biocides in air fresheners might induce cytotoxicity, and that screening these compounds using lung epithelial cells may contribute to hazard assessment.

  6. Renal epithelial cells can release ATP by vesicular fusion

    Directory of Open Access Journals (Sweden)

    Randi G Bjaelde

    2013-09-01

    Full Text Available Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30, which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1 cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin reduced both the spontaneous and hypotonically (80%-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1 and vesicular transport (nocodazole. These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ∼90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50% or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8% and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells.

  7. AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Tomoyo Yoshinaga

    Full Text Available Epithelial-mesenchymal transition (EMT of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1 and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1 and an agonist for the G protein-coupled receptor 55 (GRP55, the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

  8. Sodium selectivity of semicircular canal duct epithelial cells

    Directory of Open Access Journals (Sweden)

    Harbidge Donald G

    2011-09-01

    Full Text Available Abstract Background Sodium absorption by semicircular canal duct (SCCD epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na+ under control of a glucocorticoid hormone (dexamethasone and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC, comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na+ or also K+ through an amiloride-sensitive pathway. Parasensory K+ absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. Hear.Res. doi:10.1016/j.heares.2011.05.003, 2011]. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197, whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na+ but not K+, but the Na+ selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na+. Conclusions These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na+-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na+ via the amiloride-sensitive pathway and do not provide a parasensory K+ efflux from the

  9. Mesenchymal Stromal Cell-Derived Interleukin-6 Promotes Epithelial-Mesenchymal Transition and Acquisition of Epithelial Stem-Like Cell Properties in Ameloblastoma Epithelial Cells.

    Science.gov (United States)

    Jiang, Chunmiao; Zhang, Qunzhou; Shanti, Rabie M; Shi, Shihong; Chang, Ting-Han; Carrasco, Lee; Alawi, Faizan; Le, Anh D

    2017-09-01

    Epithelial-mesenchymal transition (EMT), a biological process associated with cancer stem-like or cancer-initiating cell formation, contributes to the invasiveness, metastasis, drug resistance, and recurrence of the malignant tumors; it remains to be determined whether similar processes contribute to the pathogenesis and progression of ameloblastoma (AM), a benign but locally invasive odontogenic neoplasm. Here, we demonstrated that EMT- and stem cell-related genes were expressed in the epithelial islands of the most common histologic variant subtype, the follicular AM. Our results revealed elevated interleukin (IL)-6 signals that were differentially expressed in the stromal compartment of the follicular AM. To explore the stromal effect on tumor pathogenesis, we isolated and characterized both mesenchymal stromal cells (AM-MSCs) and epithelial cells (AM-EpiCs) from follicular AM and demonstrated that, in in vitro culture, AM-MSCs secreted a significantly higher level of IL-6 as compared to the counterpart AM-EpiCs. Furthermore, both in vitro and in vivo studies revealed that exogenous and AM-MSC-derived IL-6 induced the expression of EMT- and stem cell-related genes in AM-EpiCs, whereas such effects were significantly abrogated either by a specific inhibitor of STAT3 or ERK1/2, or by knockdown of Slug gene expression. These findings suggest that AM-MSC-derived IL-6 promotes tumor-stem like cell formation by inducing EMT process in AM-EpiCs through STAT3 and ERK1/2-mediated signaling pathways, implying a role in the etiology and progression of the benign but locally invasive neoplasm. Stem Cells 2017;35:2083-2094. © 2017 AlphaMed Press.

  10. Parietal epithelial cells: their role in health and disease.

    Science.gov (United States)

    Romagnani, Paola

    2011-01-01

    Parietal epithelial cells of Bowman's capsules were first described by Sir William Bowman in 1842 in his paper On the Structure and Use of the Malpighian Bodies of the Kidney [London, Taylor, 1842], but since then their functions have remained poorly understood. A large body of evidence has recently suggested that parietal epithelial cells represent a reservoir of renal progenitors in adult human kidney which generate novel podocytes during childhood and adolescence, and can regenerate injured podocytes. The discovery that parietal epithelial cells represent a potential source for podocyte regeneration suggests that podocyte injury can be repaired. However, recent results also suggest that an abnormal proliferative response of renal progenitors to podocyte injury can generate hyperplastic glomerular lesions that are observed in crescentic glomerulonephritis and other types of glomerular disorders. Taken together, these results establish an entirely novel view that changes the way of thinking about renal physiology and pathophysiology, and suggest that understanding how self-renewal and fate decision of parietal epithelial cells in response to podocyte injury may be perturbed or modulated will be crucial for obtaining novel tools for prevention and treatment of glomerulosclerosis. Copyright © 2011 S. Karger AG, Basel.

  11. Subtotal ablation of parietal epithelial cells induces crescent formation.

    NARCIS (Netherlands)

    Sicking, E.M.; Fuss, A.; Uhlig, S.; Jirak, P.; Dijkman, H.; Wetzels, J.; Engel, D.R.; Urzynicok, T.; Heidenreich, S.; Kriz, W.; Kurts, C.; Ostendorf, T.; Floege, J.; Smeets, B.; Moeller, M.J.

    2012-01-01

    Parietal epithelial cells (PECs) of the renal glomerulus contribute to the formation of both cellular crescents in rapidly progressive GN and sclerotic lesions in FSGS. Subtotal transgenic ablation of podocytes induces FSGS but the effect of specific ablation of PECs is unknown. Here, we established

  12. Metabolic cooperativity between epithelial cells and adipocytes of mice

    International Nuclear Information System (INIS)

    Bartley, J.C.; Emerman, J.T.; Bissell, M.J.

    1981-01-01

    We have demonstrated that glycogen and lipid synthesis in adipocytes is modulated by the lactational state and that this modulation in mammary adipocytes requires the presence of the adjacent epithelial cells. Glycogen and lipid synthesis from [ 14 C]glucose was measured in mammary fat pads cleared of epithelium, in abdominal fat pads, and in adipocytes from both sources and from intact mammary gland of mature virgin, pregnant, and lactating mice. Accumulation of glycogen, the activity of glycogen synthase, and the lipogenic rate in abdominal and mammary adipocytes remained high during pregnancy but decreased to insignificant levels by early lactation. The depressant effects of lactation were observed solely in those mammary adipocytes isolated from intact glands. The presence of mammary epithelial cells was also required to effect the stimulated lipogenesis in mammary adipocytes during pregnancy. We conclude that the metabolic activity of adipocytes is modulated both during pregnancy and lactation to channel nutrients to the mammary epithelial cell. The fact that the changes occur in mammary adipocytes only when epithelial cells are present indicates that local as well as systemic factors are operating in these modulations

  13. Preliminary findings on vaginal epithelial cells and body ...

    African Journals Online (AJOL)

    Dr Gatsing

    http://indexmedicus.afro.who.int. Preliminary findings on vaginal epithelial cells and body temperature changes during oestrous cycle in Bororo zebu cow. J. P. Kilekoung MINGOAS 1* and L. Lalaud NGAYAM 2. 1 School of Medicine and Veterinary Sciences, University of Ngaoundere, P.O. Box 454 Ngaoundere,. Cameroon ...

  14. Nasal epithelial cells can act as a physiological surrogate for paediatric asthma studies.

    Directory of Open Access Journals (Sweden)

    Surendran Thavagnanam

    Full Text Available INTRODUCTION: Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures. METHODS: Paired nasal and bronchial epithelial cells from asthmatic children (n = 9 were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis. RESULTS: Physiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13. CONCLUSIONS: We conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.

  15. One-year follow-up of corneal confocal microscopy after corneal cross-linking in patients with post laser in situ keratosmileusis ectasia and keratoconus.

    Science.gov (United States)

    Kymionis, George D; Diakonis, Vasilios F; Kalyvianaki, Maria; Portaliou, Dimitra; Siganos, Charalampos; Kozobolis, Vasilios P; Pallikaris, Aristophanis I

    2009-05-01

    To investigate corneal tissue alterations after corneal collagen cross-linking in patients with post laser in situ keratosmileusis (LASIK) keratectasia and keratoconus. Prospective comparative case series. Five patients (5 eyes) with iatrogenic keratectasia after LASIK and 5 patients (5 eyes) with progressive keratoconus were included. All eyes underwent corneal cross-linking and were assessed by corneal in vivo confocal microscopy. Three normal/healthy and 3 post-LASIK without ectasia corneas were also examined as controls. All corneas revealed normal epithelial thickness before and after surgery. Images of both keratoconic and post-LASIK corneal ectasia eyes revealed similar morphologic alterations. The subepithelial nerve plexus was absent immediately after treatment; regeneration of nerves was evident after the third postoperative month. Keratocytes were absent from the anterior 300 mum of the stroma in the first 3 months while the posterior stromal density of keratocytes was increased. Corneal collagen fibers in the anterior stroma were distributed unevenly in a net-like formation. Full-thickness keratocyte repopulation in the anterior and mid-corneal stroma was detected 6 months after treatment. The corneal endothelium did not undergo any significant changes, since the cell density and hexagonality was not found altered during the follow-up period. Keratocyte nuclei apoptosis in the anterior and intermediate corneal stroma along with collagen alterations were observed during the first 3 postcorneal cross-linking months. Gradual keratocyte repopulation was demonstrated over the following months. Corneal alterations after corneal cross-linking were similar in both keratoconic and post-LASIK corneal ectasia eyes.

  16. Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

    Science.gov (United States)

    Lewis, Katherine Jean Reeder

    The alveolar epithelium consists of two cell phenotypes, elongated alveolar type I cells (AT1) and rounded alveolar type II cells (ATII), and exists in a complex three-dimensional environment as a polarized cell layer attached to a thin basement membrane and enclosing a roughly spherical lumen. Closely surrounding the alveolar cysts are capillary endothelial cells as well as interstitial pulmonary fibroblasts. Many factors are thought to influence alveolar epithelial cell differentiation during lung development and wound repair, including physical and biochemical signals from the extracellular matrix (ECM), and paracrine signals from the surrounding mesenchyme. In particular, disrupted signaling between the alveolar epithelium and local fibroblasts has been implicated in the progression of several pulmonary diseases. However, given the complexity of alveolar tissue architecture and the multitude of signaling pathways involved, designing appropriate experimental platforms for this biological system has been difficult. In order to isolate key factors regulating cellular behavior, the researcher ideally should have control over biophysical properties of the ECM, as well as the ability to organize multiple cell types within the scaffold. This thesis aimed to develop a 3D synthetic hydrogel platform to control alveolar epithelial cyst formation, which could then be used to explore how extracellular cues influence cell behavior in a tissue-relevant cellular arrangement. To accomplish this, a poly(ethylene glycol) (PEG) hydrogel network containing enzymatically-degradable crosslinks and bioadhesive pendant peptides was employed as a base material for encapsulating primary alveolar epithelial cells. First, an array of microwells of various cross-sectional shapes was photopatterned into a PEG gel containing photo-labile crosslinks, and primary ATII cells were seeded into the wells to examine the role of geometric confinement on differentiation and multicellular arrangement

  17. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang, E-mail: wenfang64@hotmail.com; Zhang, Yi, E-mail: syzi960@yahoo.com

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  18. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    International Nuclear Information System (INIS)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang; Zhang, Yi

    2013-01-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients

  19. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    Science.gov (United States)

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells.

  20. Expression of S100B during the innate immune of corneal epithelium against fungi invasion

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    2016-02-01

    Full Text Available AIM: To explore the expression of S100B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro. METHODS: Immortalized human corneal epithelial cells (HCECs were exposed to inactive Aspergillus fumigatus (A. fumigatus conidia at 0, 4, 8, 12, 16, and 24h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48h and the normal rat corneas were used for normal control. The mRNA level of S100B was evaluated by real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR. S100B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining (IHC/ICC. RESULTS: Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn’t express or rarely express S100B at baseline. A. fumigatus significantly induced S100B mRNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the mRNA began to rise significantly at 8h in vitro (P<0.05 and continue to rise as time prolonged (P<0.01. In vivo, S100B mRNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12h after infection (P<0.05 and reached to a peak at 24h (P<0.001. Immunochemistry revealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100B protein. CONCLUSION: S100B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation. S100B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection.

  1. Expression of S100B during the innate immune of corneal epithelium against fungi invasion

    Science.gov (United States)

    Zhang, Jie; Zhao, Gui-Qiu; Qu, Jing; Che, Cheng-Ye; Lin, Jing; Jiang, Nan; Zhao, Han; Wang, Xue-Jun

    2016-01-01

    AIM To explore the expression of S100B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro. METHODS Immortalized human corneal epithelial cells (HCECs) were exposed to inactive Aspergillus fumigatus (A. fumigatus) conidia at 0, 4, 8, 12, 16, and 24h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48h and the normal rat corneas were used for normal control. The mRNA level of S100B was evaluated by real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). S100B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining (IHC/ICC). RESULTS Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn't express or rarely express S100B at baseline. A. fumigatus significantly induced S100B mRNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the mRNA began to rise significantly at 8h in vitro (P<0.05) and continue to rise as time prolonged (P<0.01). In vivo, S100B mRNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12h after infection (P<0.05) and reached to a peak at 24h (P<0.001). Immunochemistry revealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100B protein. CONCLUSION S100B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation. S100B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection. PMID:26949634

  2. Comparison of corneal endothelial cell measurements by two non-contact specular microscopes.

    Science.gov (United States)

    Gasser, Laura; Reinhard, Thomas; Böhringer, Daniel

    2015-07-29

    Measurement of corneal endothelial cell density is important both for clinical diagnosis as well as clinical studies. Since endothelial cell loss is considered irreversible in humans, even small changes in endothelial cell density are relevant. Therefore it is important to know whether different instruments for endothelial cell density measurements give the same results and can thus be used interchangeably. In this study we compare corneal endothelial cell density and morphometry measurements from two widely used non-contact specular microscopes, the Topcon SP3000P and Konan Noncon Robo SP8000. Endothelial cell measurements were performed with both the Topcon SP3000P and Konan Noncon Robo SP8000 on 34 eyes of 18 consecutive patients of our cornea clinics with poor image quality being the only exclusion criterion. Images were obtained using the auto-focussing method and manual cell selection. Endothelial cell density (ECD), hexagonal cell ratio (HEX) and coefficient of value (CV) of the endothelial cell layer were calculated by the instruments' built-in software. ECD values calculated by the Konan were systematically higher than Topcon values: in 94 % of eyes Konan gave a higher value than Topcon, leading to a mean difference in ECD between the instruments of 187 cells/mm(2) (P < 0.001 in paired Wilcoxon test). HEX showed a broad range of values and differed greatly with only weak correlation between the two instruments. CV values for Konan mostly exceeded Topcon values, and only showed a weak correlation between the two instruments as well. Values for ECD between the Konan and the Topcon do correlate well, but the ECDs calculated by the Konan are systematically higher than Topcon values. Both HEX and CV vary greatly and do not correlate sufficiently. Thus we recommend not to use the Konan and the Topcon instrument interchangeably.

  3. Molecular basis of potassium channels in pancreatic duct epithelial cells

    DEFF Research Database (Denmark)

    Hayashi, M.; Novak, Ivana

    2013-01-01

    Potassium channels regulate excitability, epithelial ion transport, proliferation, and apoptosis. In pancreatic ducts, K channels hyperpolarize the membrane potential and provide the driving force for anion secretion. This review focuses on the molecular candidates of functional K channels...... other cell types, preferably in epithelia, and, where known, their identification and functions in pancreatic ducts and in adenocarcinoma cells. We conclude by pointing out some outstanding questions and future directions in pancreatic K channel research with respect to the physiology of secretion...

  4. Smad2 overexpression enhances adhesion of gingival epithelial cells.

    Science.gov (United States)

    Hongo, Shoichi; Yamamoto, Tadashi; Yamashiro, Keisuke; Shimoe, Masayuki; Tomikawa, Kazuya; Ugawa, Yuki; Kochi, Shinsuke; Ideguchi, Hidetaka; Maeda, Hiroshi; Takashiba, Shogo

    2016-11-01

    Gingival epithelial cells play an important role in preventing the initiation of periodontitis, by their hemidesmosomal adhesion to the tooth root surface. Adhesion requires integrin-extracellular matrix (ECM) interactions that are intricately regulated by transforming growth factor-β (TGF-β) signaling. However, the mechanisms underlying the interplay between adhesion molecules and TGF-β, especially the respective roles of Smad2 and Smad3, remain elusive. In this study, we examined the effects of Smad overexpression on gingival epithelial cell adhesion and expression profiles of integrin and ECM-related genes. Human gingival epithelial cells immortalized by the SV40 T-antigen were transfected with Smad2- and Smad3-overexpression vectors. A cell adhesion assay involving fluorescence detection of attached cells was performed using the ArrayScan imaging system. Real-time PCR was performed to examine the kinetics of integrin and ECM gene expression. In vitro and in vivo localization of adhesion molecules was examined by immunofluorescence analysis. By using SB431542, a specific inhibitor of the TGF-β type I receptor, Smad2/3 signaling was confirmed to be dominant in TGF-β1-induced cell adhesion. The Smad2-transfectant demonstrated higher potency for cell adhesion and integrin expression (α2, α5, β4, and β6) than the Smad3-transfectant, whereas little or no change in ECM expression was observed in either transfectant. Moreover, the gingival epithelium of transgenic mice that overexpressed Smad2 driven by the keratin 14 promoter showed increased integrin α2 expression. These findings indicate the crucial role of Smad2 in increased adhesion of gingival epithelial cells via upregulation of integrin α2. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system.

    Science.gov (United States)

    Jijon, H B; Suarez-Lopez, L; Diaz, O E; Das, S; De Calisto, J; Yaffe, M B; Pittet, M J; Mora, J R; Belkaid, Y; Xavier, R J; Villablanca, E J

    2017-11-15

    Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of RA receptor α (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack of RARα resulted in increased KLF4 + goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased Reg3g, reduced luminal bacterial detection, and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.Mucosal Immunology advance online publication, 15 November 2017; doi:10.1038/mi.2017.91.

  6. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    International Nuclear Information System (INIS)

    Doupnik, C.A.; Leikauf, G.D.

    1990-01-01

    Injury to the airway mucosa after exposure to environmental irritants is associated with pulmonary inflammation and bronchial hyperresponsiveness. To better understand the relationships between mediator release and airway epithelial cell injury during irritant exposures, we studied the effects of acrolein, a low-molecular-weight aldehyde found in cigarette smoke, on arachidonic acid metabolism in cultured bovine tracheal epithelial cells. Confluent airway epithelial cell monolayers, prelabeled with [3H]arachidonic acid, released significant levels of 3H activity when exposed (20 min) to 100 microM acrolein. [3H]arachidonic acid products were resolved using reverse-phase high-performance liquid chromatography. Under control conditions the released 3H activity coeluted predominantly with the cyclooxygenase product, prostaglandin (PG) E2. After exposure to acrolein, significant peaks in 3H activity coeluted with the lipoxygenase products 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, as well as with PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. Dose-response relationships for acrolein-induced release of immunoreactive PGF2 alpha and PGE2 from unlabeled epithelial monolayers demonstrated 30 microM acrolein as the threshold dose, with 100 microM acrolein inducing nearly a fivefold increase in both PGF2 alpha and PGE2. Cellular viability after exposure to 100 microM acrolein, determined by released lactate dehydrogenase activity, was not affected until exposure periods were greater than or equal to 2 h. These results implicate the airway epithelial cell as a possible source of eicosanoids after exposure to acrolein

  7. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  8. Corneal sensation after cataract and refractive surgery.

    Science.gov (United States)

    Kohlhaas, M

    1998-10-01

    Most surgical procedures involving the anterior segment of the eye disrupt the normal organization of corneal innervation. Since denervation of the cornea results in impaired epithelial wound healing, increased epithelial permeability, decreased epithelial metabolic activity, and loss of cytoskeletal structures associated with cellular adhesion, it is important to identify the factors that determine the extent of neural regeneration. Mechanisms of corneal nerve damage and studies of corneal nerve fiber loss and reinnervation after cataract and refractive surgery--epikeratophakia, cryokeratomileusis, keratomileusis in situ, photorefractive keratectomy, laser in situ keratomileusis, and phacoemulsification--are reviewed and the decrease in corneal sensitivity, as a measure of corneal destruction and corneal metabolism, after these surgical procedures is compared.

  9. Invasion of Human Oral Epithelial Cells by Prevotella intermedia

    Science.gov (United States)

    Dorn, Brian R.; Leung, K.-P.; Progulske-Fox, Ann

    1998-01-01

    Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion. PMID:9826397

  10. Differential effects of hypoxic stress in alveolar epithelial cells and microvascular endothelial cells

    NARCIS (Netherlands)

    Signorelli, Sara; Jennings, Paul; Leonard, Martin O; Pfaller, Walter

    2010-01-01

    Under hypoxic conditions eukaryotic cells and tissues undergo adaptive responses involving glycolysis, angiogenesis, vasoconstriction and inflammation. The underlying molecular mechanisms are not yet fully elucidated and are most likely cell and tissue specific. In the lung, alveolar epithelial

  11. Morphology and neurochemistry of canine corneal innervation.

    Science.gov (United States)

    Marfurt, C F; Murphy, C J; Florczak, J L

    2001-09-01

    To determine the architectural pattern and neuropeptide content of canine corneal innervation. Corneal nerve fibers in normal dog eyes were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), and tyrosine hydroxylase (TH). Relative innervation densities and distribution patterns for each fiber population were assessed qualitatively by serial line-drawing reconstructions and quantitatively by computer-assisted analyses. More than 99% of all corneal PGP-9.5-immunoreactive (IR) nerves contained both CGRP and SP, approximately 30% contained TH, and none contained VIP. Distribution patterns of corneal PGP-9.5-, CGRP-, SP-, and TH-IR nerves were indistinguishable, except that TH-IR fibers were absent from the corneal epithelium. Morphologically, canine corneal innervation consisted of a rich anterior stromal plexus, divided on the basis of morphologic criteria into anterior and posterior levels, and a rich epithelial innervation, characterized by large numbers of horizontally oriented, basal epithelial "leash" formations. Leash axons in all quadrants of the corneal epithelium oriented preferentially toward a common locus in the perilimbal cornea. The results of this study demonstrate for the first time the detailed architectural features, distinctive basal epithelial leash orientations, and peptidergic content of canine corneal innervation. The normal innervation pattern described in this study will provide other investigators with essential baseline data for assessing corneal nerve alterations in canine patients with spontaneous chronic corneal epithelial defects (SCCED) and other ocular diseases or injuries.

  12. Safety outcomes and long-term effectiveness of ex vivo autologous cultured limbal epithelial transplantation for limbal stem cell deficiency.

    Science.gov (United States)

    Fasolo, Adriano; Pedrotti, Emilio; Passilongo, Mattia; Marchini, Giorgio; Monterosso, Cristina; Zampini, Roberto; Bohm, Elisabetta; Birattari, Federica; Franch, Antonella; Barbaro, Vanessa; Bertolin, Marina; Breda, Claudia; Di Iorio, Enzo; Ferrari, Barbara; Ferrari, Stefano; Meneguzzi, Mauro; Ponzin, Diego

    2017-05-01

    To evaluate the safety and effectiveness of ex vivo autologous cultured limbal stem cell transplantation (CLET). We reviewed the clinical records of 59 consecutive patients treated with 65 CLETs. Efficacy was graded 1 year after surgery as successful, partially successful or failed. A safety analysis was performed considering side effects and complications that were recorded during the first year after CLET and those reported later than 1 year, including the events related to subsequent treatments. The mean post-CLET follow-up was 6.0±4.1 years. 69% of CLETs had either one or more adverse events (AEs), or adverse drug reactions (ADRs), within 1 year of surgery, with inflammation being the most common (42%), followed by corneal epithelium defects/disepithelialisation (31%), and blood coagula under the fibrin (24%). One year after surgery, 41% of the 59 primary CLET procedures were successful, 39% partially successful and 20% failed. The most common ADRs recorded for the primary unsuccessful CLETs were ulcerative keratitis, melting/perforation, and epithelial defects/disepithelialisation. Six failed CLETs required reconstructive penetrating keratoplasty (PK). Among CLETs with a favourable outcome, 13 underwent corrective PK (mean 4.8±3.4 years), and thereafter seven eyes maintained integrity of the corneal epithelium, five showed corneal surface failure, and one had recurrent epithelial defects. Corneal graft rejection episodes were reported in 71% and 58% of patients following corrective or reconstructive PK, respectively. Seven primary CLETs with a favourable outcome worsened thereafter, and the overall 3-year long-term effectiveness was 68%. This study addresses important issues regarding possible risks associated with disarray of the ocular surface homeostasis following autologous CLET in patients with limbal stem cell deficiency, despite the fact that the majority of patients experienced a favourable long-term benefit. Published by the BMJ Publishing

  13. 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.

  14. 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.

  15. Human airway xenograft models of epithelial cell regeneration

    Directory of Open Access Journals (Sweden)

    Puchelle Edith

    2000-10-01

    Full Text Available Abstract Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa.

  16. Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin; Yamada, Masakazu; Akune, Yoko; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo; Tsubota, Kazuo

    2010-08-01

    The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.

  17. 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......-ethylhexyl phthalate (DEHP), diisodecyl phthalate (DIDP), di-n-octyl phthalate (DnOP), and di-isononyl phthalate (DINP). Gene expression and secretion of inflammatory cytokines were evaluated after exposure to DBP. Decreased cell proliferation was observed for the phthalates DBP, BBP, and DEHP, and cell...... line B4G12 may be a potential model for inflammatory eye irritancy testing of phthalates....

  18. Stiffness nanotomography of human epithelial cancer cells

    Science.gov (United States)

    Staunton, Jack R.; Doss, Bryant L.; Gilbert, C. Michael; Kasas, Sandor; Ros, Robert

    2012-02-01

    The mechanical stiffness of individual cells is important in both cancer initiation and metastasis. We present atomic force microscopy (AFM) based nanoindentation experiments on various human mammary and esophagus cell lines covering the spectrum from normal immortalized cells to highly metastatic ones. The combination of an AFM with a confocal fluorescence lifetime imaging microscope (FLIM) in conjunction with the ability to move the sample and objective independently allow for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. This enables us to correlate the mechanical properties with the point of indentation in the FLIM image. We are using force-volume measurements as well as force indentation curves on distinct points on the cells to compare the elastic moduli of the nuclei, nucleoli, and the cytoplasm, and how they vary within and between individual cells and cell lines. Further, a detailed analysis of the force-indentation curves allows study of the cells' mechanical properties at different indentation depths and to generate 3D elasticity maps.

  19. Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

    Science.gov (United States)

    Herceg, Zdenko

    Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the

  20. In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

    Science.gov (United States)

    Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

    2015-01-01

    Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue. © 2014 International Federation for Cell Biology.

  1. L-Carnitine Reduces in Human Conjunctival Epithelial Cells Hypertonic-Induced Shrinkage through Interacting with TRPV1 Channels

    Directory of Open Access Journals (Sweden)

    Noushafarin Khajavi

    2014-08-01

    Full Text Available Background/Aims: Ocular surface health depends on conjunctival epithelial (HCjE layer integrity since it protects against pathogenic infiltration and contributes to tissue hydration maintenance. As the same increases in tear film hyperosmolarity described in dry eye disease can increase corneal epithelial transient receptor potential vanilloid type-1 (TRPV1 channel activity, we evaluated its involvement in mediating an osmoprotective effect by L-carnitine against such stress. Methods: Using siRNA gene silencing, Ca2+ imaging, planar patch-clamping and relative cell volume measurements, we determined if the protective effects of this osmolyte stem from its interaction with TRPV1. Results: TRPV1 activation by capsaicin (CAP and an increase in osmolarity to ≈ 450 mOsM both induced increases in Ca2+ levels. In contrast, blocking TRPV1 activation with capsazepine (CPZ fully reversed this response. Similarly, L-carnitine (1 mM also reduced underlying whole-cell currents. In calcein-AM loaded cells, hypertonic-induced relative cell volume shrinkage was fully blocked during exposure to L-carnitine. On the other hand, in TRPV1 gene-silenced cells, this protective effect by L-carnitine was obviated. Conclusion: The described L-carnitine osmoprotective effect is elicited through suppression of hypertonic-induced TRPV1 activation leading to increases in L-carnitine uptake through a described Na+-dependent L-carnitine transporter.

  2. Osmoregulation of chloride channels in epithelial cells

    NARCIS (Netherlands)

    C.H. Lim (Christina)

    2008-01-01

    markdownabstract__Abstract__ The plasma membrane of mammalian cells is formed by two layers of lipids (lipid bilayer), primarily phospholipids, glycolipids and cholesterol, in which many different proteins are embedded. Phospholipid consists of a glycerol backbone esterified to fatty acids

  3. Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

    Directory of Open Access Journals (Sweden)

    Ivana Viktorinová

    2017-11-01

    Full Text Available Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

  4. Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

    Science.gov (United States)

    Viktorinová, Ivana; Henry, Ian; Tomancak, Pavel

    2017-11-01

    Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

  5. Serratia marcescens is injurious to intestinal epithelial cells.

    Science.gov (United States)

    Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

    2014-01-01

    Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens.

  6. Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells.

    Science.gov (United States)

    Kaji, Yuichi; Amano, Shiro; Usui, Tomohiko; Oshika, Tetsuro; Yamashiro, Kenji; Ishida, Susumu; Suzuki, Kaori; Tanaka, Sumiyoshi; Adamis, Anthony P; Nagai, Ryoji; Horiuchi, Seiko

    2003-02-01

    The corneal endothelium is a target of the aging process. This study was undertaken to reveal the relationship between corneal endothelial cell (CEC) death and the accumulation of advanced glycation end products (AGEs), by investigating the possible mechanism of accumulation of AGE in CECs and its effects on CEC death. First, the in vivo expression of the receptor was investigated for AGE (RAGE) and galectin-3, both receptors for AGE, at both the mRNA and protein levels. Second, AGEs were added to the culture media of the cultured CECs, and the uptake of AGEs, the generation of reactive oxygen species, and the induction of apoptosis were investigated. Immunohistochemistry and RT-PCR demonstrated that both RAGE and galectin-3 were expressed in bovine CECs. After administration of AGE-modified bovine serum albumin to the culture medium, uptake of AGE was observed in the cytoplasm of the cultured bovine CECs. In addition, with increasing concentration of AGEs, the generation of reactive oxygen and the number of apoptotic cells also increased. These results show that the accumulation of AGEs in CECs induced apoptosis, in part, by increasing cellular oxidative stress. The accumulation of AGEs in the CECs of elderly patients may be involved in the loss of CECs during the aging process.

  7. Expression of semaphorin 3A in the rat corneal epithelium during wound healing

    Energy Technology Data Exchange (ETDEWEB)

    Morishige, Naoyuki [Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505 (Japan); Ko, Ji-Ae, E-mail: jiae0831@yamaguchi-u.ac.jp [Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505 (Japan); Morita, Yukiko; Nishida, Teruo [Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505 (Japan)

    2010-05-14

    The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of

  8. In Vivo Confocal Microscopic Evaluation of Corneal Endothelial Dysfunction Induced by Phacoemulcification in Rhesus Monkey Models.

    Science.gov (United States)

    Wu, Min; Hu, Zhu-lin; Sun, Xiao-mei; Dai, Jie-jie

    2016-02-01

    To observe the characteristic morphological changes of corneal endothelial dysfunction induced by phacoemulcification in rhesus monkey models under confocal microscope. The corneal endothelial dysfunction models were established by phacoemulcification power on the central corneal of 7 to 9 mm diameter in the right eyes of 4 rhesus monkeys (the modeling group). The left eyes of 4 rhesus monkeys were set as blank control group. The structural changes in different corneal layers were evaluated by slit lamp microscope and in vivo confocal microscope before surgery and 1, 2, 3, and 4 weeks after surgery. SPSS 19.0 software was applied to analyze data. Paired-t test was used to compare the number of nerve plexus in Bowman's layer and corneal endothelial cell density. Analysis of variance (ANOVA) was used to analyze corneal thickness. After phacoemulcification, the changes of cornea occurred gradually in the endothelial layer, stroma, Bowman's membrane, and basal epithelial layer. In the early stage, the interspace of corneal endothelial cells enlarged and few activated stromal cells were detected in the stroma. The cell morphology of stroma altered. The thickness of stroma increased. Two weeks after surgery, the nerve plexus in Bowman's layer decreased and edema of stroma and endothelial layer increased. Three weeks after surgery, the interspace of basal epithelial cells increased with a few Langerhans' cells infiltration and edema of stroma and endothelial layer increased. Four weeks after the surgery, a large amount of Langerhans' cells presented in basal epithelial layer. Only a few nerve lexus could be seen in Bowman's layer. The stroma and endothelial cells had severe edema. A large number of activated stromal cells could be found in stromal layer. Two weeks after the surgery, the number of nerve plexus in Bowman's layer (t=6.9192, P=0.002) and corneal endothelial cell density (t=7.8936, P<0.0001) in the modeling group were significantly lower than that in control

  9. γδ T cells in homeostasis and host defence of epithelial barrier tissues

    DEFF Research Database (Denmark)

    Nielsen, Morten M.; Witherden, Deborah A.; Havran, Wendy L.

    2017-01-01

    Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue...

  10. Keratocyte loss in corneal infection through apoptosis: a histologic study of 59 cases

    Directory of Open Access Journals (Sweden)

    Garg Prashant

    2004-12-01

    Full Text Available Abstract 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 Labelling (TUNEL staining on 59 corneal buttons from patients diagnosed as bacterial, fungal, viral and Acanthamoeba keratitis. The corneal sections were reviewed for morphologic changes in the epithelium, stroma, type, degree and depth of inflammation, loss of keratocytes in the surrounding stroma (posterior or peripheral. TUNEL positivity was evaluated in the corneal sections, both in the zone of inflammation as well as the surrounding stroma. A correlation was attempted between the keratocyte loss, histologic, microbiologic and clinical features. Results The corneal tissues were from 59 patients aged between 16 years and 85 years (mean 46 years and included fungal (22, viral (15, bacterial (14 and Acanthamoeba (8 keratitis. The morphological changes in corneal tissues noted were: epithelial ulceration (52, 88.1%, destruction of Bowman's layer (58, 99%, mild to moderate (28; 47.5% to severe inflammation (31; 52.5%. Morphologic evidence of disappearance or reduced number of keratocytic nuclei in the corneal stroma was noted in 49 (83% cases; while the TUNEL positive brown cells were identified in all cases 53/54 (98%, including cases of fungal (19, bacterial (14, viral (13, and Acanthamoeba keratitis. TUNEL staining was located mostly in the deeper stroma

  11. Erythropoietin Induces an Epithelial to Mesenchymal Transition-Like Process in Mammary Epithelial Cells MCF10A.

    Science.gov (United States)

    Ordoñez-Moreno, Alejandra; Rodriguez-Monterrosas, Cecilia; Cortes-Reynosa, Pedro; Perez-Carreon, Julio Isael; Perez Salazar, Eduardo

    2017-09-01

    Anemia is associated with chemotherapy treatment in cancer patients. Erythropoietin (EPO) has been used to treat anemia of cancer patients, because it stimulates erythropoiesis. However, treatment of breast cancer patients with EPO has been associated with poor prognosis and decrease of survival. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells are transdifferentiated to a mesenchymal state. It has been implicated in tumor progression, because epithelial cells acquire the capacity to execute the multiple steps of invasion/metastasis process. However, the role of EPO on EMT process in human mammary epithelial cells has not been studied. In the present study, we demonstrate that EPO promotes a decrease of E-cadherin expression, an increase of N-cadherin, vimentin, and Snail2 expression, activation of FAK and Src kinases and an increase of MMP-2 and MMP-9 secretions. Moreover, EPO induces an increase of NFκB DNA binding activity, an increase of binding of p50 and p65 NFκB subunits to Snail1 promoter, migration, and invasion in mammary non-tumorigenic epithelial cells MCF10A. In summary, these findings demonstrate, for the first time, that EPO induces an EMT-like process in mammary non-tumorigenic epithelial cells. J. Cell. Biochem. 118: 2983-2992, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Extended latanoprost release from commercial contact lenses: in vitro studies using corneal models.

    Directory of Open Access Journals (Sweden)

    Saman Mohammadi

    Full Text Available In this study, we compared, for the first time, the release of a 432 kDa prostaglandin F2a analogue drug, Latanoprost, from commercially available contact lenses using in vitro models with corneal epithelial cells. Conventional polyHEMA-based and silicone hydrogel soft contact lenses were soaked in drug solution (131 μg = ml solution in phosphate buffered saline. The drug release from the contact lens material and its diffusion through three in vitro models was studied. The three in vitro models consisted of a polyethylene terephthalate (PET membrane without corneal epithelial cells, a PET membrane with a monolayer of human corneal epithelial cells (HCEC, and a PET membrane with stratified HCEC. In the cell-based in vitro corneal epithelium models, a zero order release was obtained with the silicone hydrogel materials (linear for the duration of the experiment whereby, after 48 hours, between 4 to 6 μg of latanoprost (an amount well within the range of the prescribed daily dose for glaucoma patients was released. In the absence of cells, a significantly lower amount of drug, between 0.3 to 0.5 μg, was released, (p <0:001. The difference observed in release from the hydrogel lens materials in the presence and absence of cells emphasizes the importance of using an in vitro corneal model that is more representative of the physiological conditions in the eye to more adequately characterize ophthalmic drug delivery materials. Our results demonstrate how in vitro models with corneal epithelial cells may allow better prediction of in vivo release. It also highlights the potential of drug-soaked silicone hydrogel contact lens materials for drug delivery purposes.

  13. [Adhesion of clinical Candida albicans isolate to buccal epithelial cells].

    Science.gov (United States)

    Wellmer, A

    1999-01-01

    Mucosal adherence and germ tube formation are considered to be important virulence factors of C. albicans. Adherence is a precondition for colonisation and invasion. We investigated 11 clinical isolates (among them 5 cases recovered from oesophageal thrush) for quantification of the two characteristics and correlated the results with clinical data. Adherence was measured on buccal epithelial cells and the continuous flow culture was used for quantification of germ tube formation. Adherence of strains recovered from clinically, culturally and serologically confirmed oesophageal thrush adhered stronger to buccal epithelial cells than isolates from patients with heavy colonisation without signs of candidosis. Strains with stronger adherence showed a significantly faster and an increased germ tube formation in the continuous flow culture. Strains from oesophageal thrush therefore show a more marked expression of the investigated virulence factors. Therefore a good adherence is a necessity for infection of the oesophagus by C. albicans. The preferential isolation of C. albicans from oesophageal thrush (> 90%) supports this assumption.

  14. Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine

    NARCIS (Netherlands)

    Klunder, Leon J.; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C. D.

    Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we

  15. Cytotoxicity of atropine to human corneal endothelial cells by inducing mitochondrion-dependent apoptosis.

    Science.gov (United States)

    Wen, Qian; Fan, Ting-Jun; Tian, Cheng-Lei

    2016-07-01

    Atropine, a widely used topical anticholinergic drug, might have adverse effects on human corneas in vivo. However, its cytotoxic effect on human corneal endothelium (HCE) and its possible mechanisms are unclear. Here, we investigated the cytotoxicity of atropine and its underlying cellular and molecular mechanisms using an in vitro model of HCE cells and verified the cytotoxicity using cat corneal endothelium (CCE) in vivo. Our results showed that atropine at concentrations above 0.3125 g/L could induce abnormal morphology and viability decline in a dose- and time-dependent manner in vitro. The cytotoxicity of atropine was proven by the induced density decrease and abnormality of morphology and ultrastructure of CCE cells in vivo. Meanwhile, atropine could also induce dose- and time-dependent elevation of plasma membrane permeability, G1 phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCE cells. Moreover, 2.5 g/L atropine could also induce caspase-2/-3/-9 activation, mitochondrial transmembrane potential disruption, downregulation of anti-apoptotic Bcl-2 and Bcl-xL, upregulation of pro-apoptotic Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. In conclusion, atropine above 1/128 of its clinical therapeutic dosage has a dose- and time-dependent cytotoxicity to HCE cells in vitro which is confirmed by CCE cells in vivo, and its cytotoxicity is achieved by inducing HCE cell apoptosis via a death receptor-mediated mitochondrion-dependent signaling pathway. Our findings provide new insights into the cytotoxicity and apoptosis-inducing effect of atropine which should be used with great caution in eye clinic. © 2016 by the Society for Experimental Biology and Medicine.

  16. MAST CELLS AND ANGIOGENESIS IN ORAL EPITHELIAL DYSPLASTIC LESIONS AND ORAL SQUAMOUS CELL CARCINOMA

    OpenAIRE

    Veda, Marla Vinay

    2015-01-01

    Background: The progression of oral epithelial dysplastic lesions into oral squamous cell carcinoma is characterized by an ‘angiogenic switch’ which is characterized by an increase in neo-vascularization in the sub-epithelial lamina propria which can be considered an indicator of malignant transformation. Mast cells are a rich source of various angiogenic factors. Moreover mast cells secrete various proteolytic enzymes which degrade the extracellular matrix and create space for the developing...

  17. Fusion of intestinal epithelial cells with bone marrow derived cells is dispensable for tissue homeostasis

    OpenAIRE

    de Jong, Joan H.; Rodermond, Hans M.; Zimberlin, Cheryl D.; Lascano, Valeria; De Sousa E Melo, Felipe; Richel, Dick J.; Medema, Jan Paul; Vermeulen, Louis

    2012-01-01

    The epithelial lining of the intestine is characterized by an immense cellular turn-over ascertaining an extensive regenerative capacity. Multiple reports suggest that besides the local intestinal stem cell pool, circulating cells of bone marrow origin (BMDCs) contribute to this process by fusing with the epithelial lineage. However, the functional relevance of these observations is unknown. In the present study we employ a model system in which we cannot only detect cell fusion but also exam...

  18. Expression of ICAM-1 in colon epithelial cells

    DEFF Research Database (Denmark)

    Vainer, Ben; Sørensen, Susanne; Seidelin, Jakob

    2003-01-01

    Studies have suggested that in ulcerative colitis (UC), intercellular adhesion molecule-1 (ICAM-1) is involved in migration of leukocytes toward the colonic epithelium. A suitable in vitro model of chronic colonic inflammation does not exist, and the role of the epithelium is based on monolayers...... of cancer cells. Conflicting results exist on epithelial ICAM-1 expression, and the aim of this study was to compare the expression in various models of colonic epithelium....

  19. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducibl...... nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells....

  20. Attachment of Giardia lamblia to rat intestinal epithelial cells.

    OpenAIRE

    Inge, P M; Edson, C M; Farthing, M J

    1988-01-01

    The human enteric protozoan, Giardia lamblia, has surface membrane lectin activity which mediates parasite adherence to erythrocytes. To determine whether an intestinal binding site exists for this lectin we have studied the interaction in vitro between axenically cultured Giardia trophozoites and isolated rat intestinal epithelial cells. Scanning electron microscopy showed that Giardia attached to the apical microvillus membrane and basolateral membrane of rat enterocytes. Any location on th...

  1. Serum-Free Cryopreservation of Human Amniotic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    H. Niknejad

    2013-04-01

    Full Text Available Introduction & Objective: One of the important issues in long term storage of cells is removal of animal serum from cell culture environments. The aim of this study was to evaluate amni-otic fluid (AF, which is full of growth factors, as substitute for fetal bovine serum (FBS in the cryopreservation protocol. Materials & Methods: In this experimental study human amniotic epithelial cells were isolated from placentas which were seronegative for microbial infections. The cells were preserved in 24 different patterns for 12 months in -196 ?C (liquid nitrogen and viability of cells were determined before and after cryopreservation by trypan blue and MTT assay. Moreover, Oct-4 expression was studied to determine pluripotency before and after cryopreservation with immunocytochemistry. Results were compared between groups with ANOVA (Tukey Post-Test. P.value under 0.01 and 0.05 was considered statistically significant. Results: The presence of DMEM, FBS or AF is necessary for amniotic cell cryopreservation. Trypan-blue, MTT and immunocytochemistry showed that there isn’t significant difference between using AF and FBS in viability and pluripotency of cells. Moreover, results showed that DMSO is a better cryoprotectant compared to glycerol. Conclusion : Results showed that amniotic fluid can be a proper substitute for FBS in amniotic epithelial cells cryopreservation. (Sci J Hamadan Univ Med Sci 2013; 20 (1:15-24

  2. TCDD alters medial epithelial cell differentiation during palatogenesis

    International Nuclear Information System (INIS)

    Abbott, B.D.; Birnbaum, L.S.

    1989-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely distributed, persistent environmental contaminant that is teratogenic in mice, where it induces hydronephrosis and cleft palate. The incidence of clefting has been shown to be dose dependent after exposure on either gestation Day (GD) 10 or 12, although the embryo is more susceptible on GD 12. TCDD-exposed palatal shelves meet but do not fuse, and programmed cell death of the medial epithelial cells is inhibited. The mechanism of action through which TCDD alters the program of medial cell development has not been examined in earlier studies, and it is not known whether the mechanism is the same regardless of the dose or developmental stage of exposure. In this study, C57BL/6N mice, a strain sensitive to TCDD, were dosed orally on GD 10 or 12 with 0, 6, 12, 24, or 30 micrograms/kg body wt, in 10 ml corn oil/kg. Embryonic palatal shelves were examined on GD 14, 15, or 16. The degree of palatal closure, epithelial surface morphology, and cellular ultrastructure, the incorporation of [3H]TdR, the expression of EGF receptors, and the binding of 125I-EGF were assessed. After exposure on GD 10 or 12, TCDD altered the differentiation pathway of the medial epithelial cells. The palatal shelves were of normal size and overall morphology, but fusion of the medial epithelia of the opposing shelves did not occur. TCDD prevented programmed cell death of the medial peridermal cells. The expression of EGF receptors by medial cells continued through Day 16 and the receptors were able to bind ligand. The medial cells differentiated into a stratified, squamous, keratinizing epithelium. The shift in phenotype to an oral-like epithelium occurred after exposure on either GD 10 or 12. At the lower dose (6 micrograms/kg), fewer cleft palates were produced, but those shelves which did respond had a fully expressed shift in differentiation

  3. Isolation, separation, and characterization of epithelial and connective cells from rat palate

    Energy Technology Data Exchange (ETDEWEB)

    Terranova, Victor Paul [Univ. of Rochester, NY (United States)

    1979-01-01

    Epithelial and connective tissue cells were isolated from rat palate by sequential collagenase, hyaluronidase and trypsin digestion of the extracellular matrix. Differences between the two populations were noted with respect to total cell protein, total cell water, proline uptake and incorporation, percent collagen synthesized, effects of parathyroid hormone, metabolism of D-valine and cell density. Basal epithelial cells were subsequently separated from the heterogeneous epithelial cell population on shallow linear density gradients by velocity centrifugation. The type of collagen synthesized by the basal epithelial cells was compared to the type of collagen synthesized by the connective tissue cells by means of labeled amino acid incorporation ratios. Cells isolated from the epithelial and connective tissue were compared. From these studies it can be concluded that epithelial and connective tissue cells can be isolated from rat palate as viable and distinct populations with respect to the biochemical parameters examined. Furthermore, subpopulations can be separated and biochemically characterized.

  4. Airway Epithelial Cell Cilia and Obstructive Lung Disease

    Directory of Open Access Journals (Sweden)

    Asma Yaghi

    2016-11-01

    Full Text Available Airway epithelium is the first line of defense against exposure of the airway and lung to various inflammatory stimuli. Ciliary beating of airway epithelial cells constitutes an important part of the mucociliary transport apparatus. To be effective in transporting secretions out of the lung, the mucociliary transport apparatus must exhibit a cohesive beating of all ciliated epithelial cells that line the upper and lower respiratory tract. Cilia function can be modulated by exposures to endogenous and exogenous factors and by the viscosity of the mucus lining the epithelium. Cilia function is impaired in lung diseases such as COPD and asthma, and pharmacologic agents can modulate cilia function and mucus viscosity. Cilia beating is reduced in COPD, however, more research is needed to determine the structural-functional regulation of ciliary beating via all signaling pathways and how this might relate to the initiation or progression of obstructive lung diseases. Additionally, genotypes and how these can influence phenotypes and epithelial cell cilia function and structure should be taken into consideration in future investigations.

  5. Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro

    International Nuclear Information System (INIS)

    Moore, Jennifer S.; Rahemtulla, Firoz; Kent, Leigh W.; Hall, Stacy D.; Ikizler, Mine R.; Wright, Peter F.; Nguyen, Huan H.; Jackson, Susan

    2003-01-01

    Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity

  6. The effects of dexamethasone on the Na,K-ATPase activity and pump function of corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin; Yamada, Masakazu; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo

    2009-05-01

    The Na(+)- and K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the possible role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to dexamethasone. ATPase activity of the cells was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with the use of an Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis was examined to measure the expression of the Na,K-ATPase alpha(1)-subunit. Dexamethasone (1 or 10 microM) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of dexamethasone were blocked by cycloheximide, a protein synthesis inhibitor. Western blot analysis also indicated that dexamethasone increased the expression of the Na,K-ATPase alpha(1)-subunit, whereas it decreased the expression of the phospho-Na,K-ATPase alpha(1)-subunit. Our results suggest that dexamethasone stimulates Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells is mediated by Na,K-ATPase synthesis and increase in an enzymatic activity by dephosphorylation of Na,K-ATPase alpha(1)-subunits.

  7. Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation

    NARCIS (Netherlands)

    Loerke, D.; le Duc, Q.; Blonk, I.; Kerstens, A.; Spanjaard, E.; Machacek, M.; Danuser, G.; de Rooij, J.

    2012-01-01

    The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that

  8. Transcriptional profiling of putative human epithelial stem cells.

    Science.gov (United States)

    Koçer, Salih S; Djurić, Petar M; Bugallo, Mónica F; Simon, Sanford R; Matic, Maja

    2008-07-30

    comprehensive gene expression profile of putative human epithelial stem cells and their progeny that were isolated directly from neonatal foreskin tissue. Our study is important for understanding self renewal and differentiation of epidermal stem cells, and for elucidating signaling pathways involved in those processes. The generated data base may serve those working with other human epithelial tissue progenitors.

  9. Transcriptional profiling of putative human epithelial stem cells

    Directory of Open Access Journals (Sweden)

    Koçer Salih S

    2008-07-01

    may be enriched for stem cells. This study is the first comprehensive gene expression profile of putative human epithelial stem cells and their progeny that were isolated directly from neonatal foreskin tissue. Our study is important for understanding self renewal and differentiation of epidermal stem cells, and for elucidating signaling pathways involved in those processes. The generated data base may serve those working with other human epithelial tissue progenitors.

  10. Adherence of oral streptococci to keratinized and nonkeratinized human oral epithelial cells.

    OpenAIRE

    Sklavounou, A; Germaine, G R

    1980-01-01

    The ability of Streptococcus mutans, Streptococcus sanguis, Streptococcus mitis, and Streptococcus salivarius to adhere to keratinized versus nonkeratinized human oral epithelial cells was compared. S. mitis and S. salivarius exhibited significantly greater adherence to keratinized cells than to nonkeratinized cells. S. mutans and S. sanguis adhered equally well to either epithelial cell type. It is concluded that keratinization of epithelial cells may be a significant factor in the adherence...

  11. Generation of Breast Cancer Stem Cells through Epithelial-Mesenchymal Transition

    OpenAIRE

    Morel, Anne-Pierre; Lièvre, Marjory; Thomas, Clémence; Hinkal, George; Ansieau, Stéphane; Puisieux, Alain

    2008-01-01

    Recently, two novel concepts have emerged in cancer biology: the role of so-called "cancer stem cells" in tumor initiation, and the involvement of an epithelial-mesenchymal transition (EMT) in the metastatic dissemination of epithelial cancer cells. Using a mammary tumor progression model, we show that cells possessing both stem and tumorigenic characteristics of "cancer stem cells" can be derived from human mammary epithelial cells following the activation of the Ras-MAPK pathway. The acquis...

  12. Prion infection of epithelial Rov cells is a polarized event.

    Science.gov (United States)

    Paquet, Sophie; Sabuncu, Elifsu; Delaunay, Jean-Louis; Laude, Hubert; Vilette, Didier

    2004-07-01

    During prion infections, the cellular glycosylphosphatidylinositol-anchored glycoprotein PrP is converted into a conformational isoform. This abnormal conformer is thought to recruit and convert the normal cellular PrP into a likeness of itself and is proposed to be the infectious agent. We investigated the distribution of the PrP protein on the surface of Rov cells, an epithelial cell line highly permissive to prion multiplication, and we found that PrP is primarily expressed on the apical side. We further show that prion transmission to Rov cells is much more efficient if infectivity contacts the apical side, indicating that the apical and basolateral sides of Rov cells are not equally competent for prion infection and adding prions to the list of the conventional infectious agents (viruses and bacteria) that infect epithelial cells in a polarized manner. These data raise the possibility that apically expressed PrP may be involved in this polarized process of infection. This would add further support for a crucial role of PrP at the cell surface in prion infection of target cells.

  13. A pulse-dose topical 1% 5-fluorouracil treatment regimen in a young dog with corneal squamous cell carcinoma.

    Science.gov (United States)

    Overton, Taryn L; Allbaugh, Rachel A; Whitley, David; Ben-Shlomo, Gil; Griggs, Angel; Tofflemire, Kyle L; Whitley, Elizabeth M

    2015-07-01

    To describe the use of a pulse-dose topical 5-fluorouracil (5-FU) treatment regimen in a Pug dog with corneal squamous cell carcinoma (SCC). A 1-year-old, spayed female Pug was evaluated for a corneal perforation of the right eye, which was surgically stabilized with a conjunctival pedicle graft. At the time of medial canthoplasty 7 weeks later, two areas of gray-white discoloration had developed medial and lateral to the graft. Biopsy samples were obtained via superficial keratectomy while under general anesthesia. Definitive diagnosis of corneal SCC was made through histopathological examination of the surgical biopsies. Thoracic radiography and submandibular lymph node cytology revealed no evidence of metastatic neoplasia. Following healing of the corneal biopsy sites, topical 1% 5-FU ointment was applied four times daily for four consecutive days once a month, for six treatment cycles. Twenty-three months after diagnosis, the patient remains visual and comfortable with no evidence of SCC recurrence. Long-term therapy with once daily topical 1% cyclosporine solution was used to manage corneal pigmentation bilaterally. The pulse-therapy 1% 5-FU protocol was a successful, convenient, and cost-effective adjunctive treatment with few adverse effects. © 2014 American College of Veterinary Ophthalmologists.

  14. Cooperation between epithelial cells demonstrated by potassium transfer

    International Nuclear Information System (INIS)

    Ledbetter, M.L.; Young, G.J.; Wright, E.R.

    1986-01-01

    Junction-mediated communication can be measured in fibroblast cultures by determining the ability of mixed cultures of cells sensitive and resistant to ouabain to concentrate K+ in the presence of ouabain. We now report the extension of this assay procedure to cultured epithelial cells. Hamster kidney (HaK) cells maintain their ability to concentrate K+ in ouabain at levels inhibitory to dog kidney (MDCK) cells. When HaK and MDCK cells were cultured together in ouabain-containing medium, the K+ (measured as 86Rb+) in the mixed population was greater than expected if the cells were not interacting. The degree of enhancement, expressed as index of cooperation, depended on the numbers of cells in the cultures, their opportunity for cell-to-cell contact, and (above a certain permissive level) the concentration of ouabain. As with other cell types, protein synthesis in MDCK cells depends on maintenance of cell K+. Autoradiography of cells incubated with [3H]leucine demonstrated that MDCK cells in ouabain-treated mixed cultures were able to synthesize proteins only when physically adjacent to HaK cells. The transmission of labeled nucleosides among the cells provides independent evidence of the phenomenon of cooperation, probably mediated by gap junctions. This system offers promise for investigation of stimuli modulating junctional communication

  15. Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

    Science.gov (United States)

    Perruchot, Marie-Hélène; Arévalo-Turrubiarte, Magdalena; Dufreneix, Florence; Finot, Laurence; Lollivier, Vanessa; Chanat, Eric; Mayeur, Frédérique; Dessauge, Frédéric

    2016-10-01

    The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation.

  16. Adenosine opposes thrombin-induced inhibition of intercellular calcium wave in corneal endothelial cells.

    Science.gov (United States)

    D'hondt, Catheleyne; Srinivas, Sangly P; Vereecke, Johan; Himpens, Bernard

    2007-04-01

    In corneal endothelial cells, intercellular Ca(2+) waves elicited by a mechanical stimulus involve paracrine intercellular communication, mediated by ATP release via connexin hemichannels, as well as gap junctional intercellular communication. Both mechanisms are inhibited by thrombin, which activates RhoA and hence results in myosin light chain phosphorylation. This study was conducted to examine the effects of adenosine, which is known to oppose thrombin-induced RhoA activation, thereby leading to myosin light chain dephosphorylation, on gap junctional intercellular communication and paracrine intercellular communication in cultured bovine corneal endothelial cells. An intercellular Ca(2+) wave was elicited by applying a mechanical stimulus to a single cell in a confluent monolayer. The area of Ca(2+) wave propagation was measured by [Ca(2+)](i) imaging using the fluorescent dye Fluo-4. Gap junctional intercellular communication was assessed by fluorescence recovery after photobleaching. Activity of hemichannels was determined by uptake of the hydrophilic dye Lucifer yellow in a Ca(2+)-free medium containing 2 mM EGTA. Adenosine triphosphate (ATP) release in response to mechanical stimulation was measured using the luciferin-luciferase technique. Gap26, a connexin mimetic peptide, was used to block hemichannels. Exposure to thrombin or TRAP-6 (a selective PAR-1 agonist) inhibited the Ca(2+) wave propagation by 70%. Pretreatment with adenosine prevented this inhibitory effect of thrombin. NECA (a potent A2B agonist) and forskolin, agents known to elevate cAMP in bovine corneal endothelial cells, also suppressed the effect of thrombin. The A1 receptor agonist CPA failed to inhibit the effect of thrombin. Similar to the effects on Ca(2+) wave propagation, adenosine prevented the thrombin-induced reduction in the fluorescence recovery during photobleaching experiments. Furthermore, pretreatment with adenosine prevented both thrombin and TRAP-6 from blocking the

  17. Quantitative assessment of rat corneal thickness and morphology during stem cell therapy by high-speed optical coherence tomography

    Science.gov (United States)

    Lal, Cerine; McGrath, James; Subhash, Hrebesh; Rani, Sweta; Ritter, Thomas; Leahy, Martin

    2016-03-01

    Optical Coherence Tomography (OCT) is a non-invasive 3 dimensional optical imaging modality that enables high resolution cross sectional imaging in biological tissues and materials. Its high axial and lateral resolution combined with high sensitivity, imaging depth and wide field of view makes it suitable for wide variety of high resolution medical imaging applications at clinically relevant speed. With the advent of swept source lasers, the imaging speed of OCT has increased considerably in recent years. OCT has been used in ophthalmology to study dynamic changes occurring in the cornea and iris, thereby providing physiological and pathological changes that occur within the anterior segment structures such as in glaucoma, during refractive surgery, lamellar keratoplasty and corneal diseases. In this study, we assess the changes in corneal thickness in the anterior segment of the eye during wound healing process in a rat corneal burn model following stem cell therapy using high speed swept source OCT.

  18. Streptococcus equi subsp. zooepidemicus Invades and Survives in Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Bolette Skive

    2017-11-01

    Full Text Available Streptococcus equi subsp. zooepidemicus (S. zooepidemicus is an opportunistic pathogen of several species including humans. S. zooepidemicus is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently S. zooepidemicus was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the S. zooepidemicus pathogenesis and investigated if S. zooepidemicus was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two S. zooepidemicus strains (1-4a and S31A1 both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time points during the 23 h infection assay and field emission scanning electron microscopy (FESEM was used to characterize adhesion and invasion mechanisms. The FESEM images showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells indicating that attachment and invasion were active processes. Adherent and intracellular S. zooepidemicus, and bacteria in association with lysosomes was determined by immunofluorescence staining techniques and fluorescence microscopy. Quantification of intracellular bacteria was determined in penicillin protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive intracellularly, but the survival rate decreased over time in the cell culture system. Phagosome-like compartments containing S. zooepidemicus at some stages fused with

  19. Streptococcus equi subsp. zooepidemicus Invades and Survives in Epithelial Cells.

    Science.gov (United States)

    Skive, Bolette; Rohde, Manfred; Molinari, Gabriella; Braunstein, Thomas Hartig; Bojesen, Anders M

    2017-01-01

    Streptococcus equi subsp. zooepidemicus ( S. zooepidemicus ) is an opportunistic pathogen of several species including humans. S. zooepidemicus is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently S. zooepidemicus was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the S. zooepidemicus pathogenesis and investigated if S. zooepidemicus was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two S. zooepidemicus strains (1-4a and S31A1) both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time points during the 23 h infection assay and field emission scanning electron microscopy (FESEM) was used to characterize adhesion and invasion mechanisms. The FESEM images showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells indicating that attachment and invasion were active processes. Adherent and intracellular S. zooepidemicus , and bacteria in association with lysosomes was determined by immunofluorescence staining techniques and fluorescence microscopy. Quantification of intracellular bacteria was determined in penicillin protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive intracellularly, but the survival rate decreased over time in the cell culture system. Phagosome-like compartments containing S. zooepidemicus at some stages fused with lysosomes to form a

  20. Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles.

    Directory of Open Access Journals (Sweden)

    Giulia Chiabotto

    Full Text Available Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs produced by human renal proximal tubular epithelial cells (RPTECs may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs. To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the in vitro uptake and activity of EVs on MSCs. MicroRNA (miRNA analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs.

  1. Proteomic profiling of acrolein adducts in human lung epithelial cells

    Science.gov (United States)

    Spiess, Page C.; Deng, Bin; Hondal, Robert J.; Matthews, Dwight E.; van der Vliet, Albert

    2011-01-01

    Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway epithelial protein targets for Michael adduction in acrolein-exposed bronchial epithelial (HBE1) cells, based on biotin hydrazide labeling and avidin purification of biotinylated proteins or peptides for analysis by LC-MS/MS. Identified protein targets included a number of stress proteins, cytoskeletal proteins, and several key proteins involved in redox signaling, including thioredoxin reductase, thioredoxin, peroxiredoxins, and glutathione S-transferase π. Because of the central role of thioredoxin reductase in cellular redox regulation, additional LC-MS/MS characterization was performed on purified mitochondrial thioredoxin reductase to identify the specific site of acrolein adduction, revealing the catalytic selenocysteine residue as the target responsible for enzyme inactivation. Our findings indicate that these approaches are useful in characterizing major protein targets for acrolein, and will enhance mechanistic understanding of the impact of acrolein on cell biology. PMID:21704744

  2. Low Temperature Plasma for the Treatment of Epithelial Cancer Cells

    Science.gov (United States)

    Mohades, Soheila

    Biomedical applications of low temperature plasmas (LTP) may lead to a paradigm shift in treating various diseases by conducting fundamental research on the effects of LTP on cells, tissues, organisms (plants, insects, and microorganisms). This is a rapidly growing interdisciplinary research field that involves engineering, physics, life sciences, and chemistry to find novel solutions for urgent medical needs. Effects of different LTP sources have shown the anti-tumor properties of plasma exposure; however, there are still many unknowns about the interaction of plasma with eukaryotic cells which must be elucidated in order to evaluate the practical potential of plasma in cancer treatment. Plasma, the fourth state of matter, is composed of electrons, ions, reactive molecules (radicals and non-radicals), excited species, radiation, and heat. A sufficient dose (time) of plasma exposure can induce death in cancer cells. The plasma pencil is employed to study the anti-tumor properties of this treatment on epithelial cells. The plasma pencil has been previously used for the inactivation of bacteria, destroying amyloid fibrils, and the killing of various cancer cells. Bladder cancer is the 9th leading cause of cancer. In this dissertation, human urinary bladder tissue with the squamous cell carcinoma disease (SCaBER cells) is treated with LTP utilizing two different approaches: direct plasma exposure and Plasma Activated Media (PAM) as an advancement to the treatment. PAM is produced by exposing a liquid cell culture medium to the plasma pencil. Direct LTP treatment of cancer cells indicates a dose-dependent killing effect at post-treatment times. Similarly, PAM treatment shows an anti-cancer effect by inducing substantial cell death. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have an important role in the biomedical effects of LTP treatment. This study demonstrates the capability of the plasma pencil to transport ROS/RNS into cell culture media

  3. Explore the full thick layer of corneal transplantation in the treatment of pseudomonas aeruginosa corneal ulcer infection

    Directory of Open Access Journals (Sweden)

    Xin Wang

    2015-02-01

    Full Text Available AIM: To explore the feasibility, safety and effect of the full-thickness lamellar keratoplasty for the treatment of pseudomonas aeruginosa corneal ulcer. METHODS: Based on a retrospective non-controlled study, 25 patients were given the full-thickness lamellar keratoplasty for clinical diagnosis of pseudomonas aeruginosa infection and corneal ulcer medication conventional anti-gram-negative bacteria. Routine follow-up were carried out at postoperative 1wk; 1, 3, 6, 12, 18mo to observe the situation of corneal epithelial healing, recurrent infection, immune rejection, graft transparency and best corrected visual acuity, etc. At the 6 and 12mo postoperative, corneal endothelial cell density was reexamined.RESULTS: No patients because of Descemet's membrane rupture underwent penetrating keratoplasty surgery: One only in cases of bacterial infection after 1mo, once again did not cultivate a culture of bacteria pseudomonas aeruginosa, and the remaining 24 cases average follow-up 14±6mo, corneal graft were transparent, the cure rate was 96%. At the sixth month after surgery, there were 16 cases of eye surgery best corrected visual acuity ≥4.5, of which 3 cases ≥4.8. At the sixth month after surgery, the average corneal endothelial cell density 2 425±278/mm2; At 12mo postoperatively, it was 2 257± 326/mm2.CONCLUSION: Full-thickness lamellar keratoplasty is an effective method of pseudomonas aeruginosa infection in the treatment of corneal ulcers, corneal drying material glycerol can be achieved by visual effects.

  4. Native type IV collagen induces an epithelial to mesenchymal transition-like process in mammary epithelial cells MCF10A.

    Science.gov (United States)

    Espinosa Neira, Roberto; Salazar, Eduardo Perez

    2012-12-01

    Basement membrane (BM) is a complex network of interacting proteins, including type IV collagen (Col IV) that acts as a scaffold that stabilizes the physical structures of tissues and regulates cellular processes. In the mammary gland, BM is a continuous deposit that separates epithelial cells from stroma, and its degradation is related with an increased potential for invasion and metastasis. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells are transdifferentiated to one mesenchymal state, and is a normal process during embryonic development, tissue remodeling and wound healing, as well as it has been implicated during cancer progression. In breast cancer cells, native Col IV induces migration and gelatinases secretion. However, the role of native Col IV on the EMT process in human mammary epithelial cells remains to be investigated. In the present study, we demonstrate that native Col IV induces down-regulation of E-cadherin expression, accompanied with an increase of Snail1, Snail2 and Sip1 transcripts. Native Col IV also induces an increase in N-cadherin and vimentin expression, an increase of MMP-2 secretion, the activation of FAK and NFκB, cell migration and invasion in MCF10A cells. In summary, these findings demonstrate, for the first time, that native Col IV induces an EMT-like process in MCF10A human mammary non-tumorigenic epithelial cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. ATP7B detoxifies silver in ciliated airway epithelial cells

    International Nuclear Information System (INIS)

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-01-01

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B -/- mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag + /Cu + transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  6. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection

    Science.gov (United States)

    Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza...

  7. Characterization of biomaterial-free cell sheets cultured from human oral mucosal epithelial cells.

    Science.gov (United States)

    Hyun, Dong Won; Kim, Yun Hee; Koh, Ah Young; Lee, Hyun Ju; Wee, Won Ryang; Jeon, Saewha; Kim, Mee Kum

    2017-03-01

    The purpose of this study was to report the characteristics of biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal-deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony-forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki-67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial-free sheets. Biomaterial-free sheets which had been detached with 1% dispase or biomaterial-free sheets generated by fibrin support were transplanted to 12 limbal-deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial-free sheets were compared with those of sheets formed by fibrin-coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial-free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial-free sheets were comparable to those of sheets cultured by fibrin support, in limbal-deficient models. Both p63 and Ki-67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal-deficient models. Our results suggest that biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal-deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Corneal dystrophies

    Directory of Open Access Journals (Sweden)

    Klintworth Gordon K

    2009-02-01

    Full Text Available Abstract The term corneal dystrophy embraces a heterogenous group of bilateral genetically determined non-inflammatory corneal diseases that are restricted to the cornea. The designation is imprecise but remains in vogue because of its clinical value. Clinically, the corneal dystrophies can be divided into three groups based on the sole or predominant anatomical location of the abnormalities. Some affect primarily the corneal epithelium and its basement membrane or Bowman layer and the superficial corneal stroma (anterior corneal dystrophies, the corneal stroma (stromal corneal dystrophies, or Descemet membrane and the corneal endothelium (posterior corneal dystrophies. Most corneal dystrophies have no systemic manifestations and present with variable shaped corneal opacities in a clear or cloudy cornea and they affect visual acuity to different degrees. Corneal dystrophies may have a simple autosomal dominant, autosomal recessive or X-linked recessive Mendelian mode of inheritance. Different corneal dystrophies are caused by mutations in the CHST6, KRT3, KRT12, PIP5K3, SLC4A11, TACSTD2, TGFBI, and UBIAD1 genes. Knowledge about the responsible genetic mutations responsible for these disorders has led to a better understanding of their basic defect and to molecular tests for their precise diagnosis. Genes for other corneal dystrophies have been mapped to specific chromosomal loci, but have not yet been identified. As clinical manifestations widely vary with the different entities, corneal dystrophies should be suspected when corneal transparency is lost or corneal opacities occur spontaneously, particularly in both corneas, and especially in the presence of a positive family history or in the offspring of consanguineous parents. Main differential diagnoses include various causes of monoclonal gammopathy, lecithin-cholesterol-acyltransferase deficiency, Fabry disease, cystinosis, tyrosine transaminase deficiency, systemic lysosomal storage

  9. Inhibition of EV71 by curcumin in intestinal epithelial cells

    Science.gov (United States)

    Chio, Chi-Chong; Lin, Jhao-Yin

    2018-01-01

    EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections. PMID:29370243

  10. Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shuxian Jiang

    2010-03-01

    Full Text Available Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

  11. The keratin-binding protein Albatross regulates polarization of epithelial cells

    OpenAIRE

    Sugimoto, Masahiko; Inoko, Akihito; Shiromizu, Takashi; Nakayama, Masanori; Zou, Peng; Yonemura, Shigenobu; Hayashi, Yuko; Izawa, Ichiro; Sasoh, Mikio; Uji, Yukitaka; Kaibuchi, Kozo; Kiyono, Tohru; Inagaki, Masaki

    2008-01-01

    The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown o...

  12. Epithelial Cell Inflammasomes in Intestinal Immunity and Inflammation

    Directory of Open Access Journals (Sweden)

    Andrea C. Lei-Leston

    2017-09-01

    Full Text Available Pattern recognition receptors (PRR, such as NOD-like receptors (NLRs, sense conserved microbial signatures, and host danger signals leading to the coordination of appropriate immune responses. Upon activation, a subset of NLR initiate the assembly of a multimeric protein complex known as the inflammasome, which processes pro-inflammatory cytokines and mediates a specialized form of cell death known as pyroptosis. The identification of inflammasome-associated genes as inflammatory bowel disease susceptibility genes implicates a role for the inflammasome in intestinal inflammation. Despite the fact that the functional importance of inflammasomes within immune cells has been well established, the contribution of inflammasome expression in non-hematopoietic cells remains comparatively understudied. Given that intestinal epithelial cells (IEC act as a barrier between the host and the intestinal microbiota, inflammasome expression by these cells is likely important for intestinal immune homeostasis. Accumulating evidence suggests that the inflammasome plays a key role in shaping epithelial responses at the host–lumen interface with many inflammasome components highly expressed by IEC. Recent studies have exposed functional roles of IEC inflammasomes in mucosal immune defense, inflammation, and tumorigenesis. In this review, we present the main features of the predominant inflammasomes and their effector mechanisms contributing to intestinal homeostasis and inflammation. We also discuss existing controversies in the field and open questions related to their implications in disease. A comprehensive understanding of the molecular basis of intestinal inflammasome signaling could hold therapeutic potential for clinical translation.

  13. Immunoregulation by airway epithelial cells (AECs against respiratory virus infection

    Directory of Open Access Journals (Sweden)

    Yan YAN

    2017-11-01

    Full Text Available The respiratory tract is primary contact site of the body and environment, and it is ventilated by 10-20 thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbes, which contain the disease-causing pathogens. Airway epithelial cells (AECs are known to have innate sensor functions, which are similar to the "professional" immune cells, such as alveolar macrophage and sub- or intra-epithelial dendritic cells (DCs. Thus AECs are able to detect invading microbial danger including different types of respiratory viruses, and mount a potent host response, for example, activating type Ⅰ interferon signaling pathway genes. To avoid chronic inflammation and maintain the immunological homeostasis, the pulmonary system has developed intrinsic mechanisms to control local immune responses. Most recently, the role of AECs in control of local immunity has gained much attention, as 1 AECs express the pattern recognition receptors (PRRs, such as Toll-like receptors, retinoic acid inducible gene Ⅰ (RIG-I-like receptor, and so on, thus AECs are equipped to participate in innate detection of microbial encounter; 2 To keep immunological homeostasis in the respiratory tract, AECs behave not only as innate immune sensors but also as immune modulators in parallel, through modulating the sensitivity of innate immune sensing of both AECs per se and sub- or intra-epithelial immune cells; 3 Loss of modularity capacity of AECs might be involved in the development of chronic airway diseases. In present review, how the AECs act will be intensively discussed in response to respiratory viruses and modulate the local immunity through cis- and trans-factors (direct and indirect factors, as well as the consequence of impairment of this control of local immunity, in the development and exacerbation of airway diseases, such as acute and chronic rhinosinusitis. DOI: 10.11855/j.issn.0577-7402.2017.10.02

  14. Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

    Directory of Open Access Journals (Sweden)

    Djida Ghoubay-Benallaoua

    Full Text Available Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC and epithelial limbal (LSC stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8, E8 supplemented with EGF (E8+ or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

  15. Hypoxia modulates infection of epithelial cells by Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Bettina Schaible

    Full Text Available Pseudomonas aeruginosa (P. aeruginosa is an opportunistic pathogen commonly associated with lung and wound infections. Hypoxia is a frequent feature of the microenvironment of infected tissues which induces the expression of genes associated with innate immunity and inflammation in host cells primarily through the activation of the hypoxia-inducible factor (HIF and Nuclear factor kappaB (NF-κB pathways which are regulated by oxygen-dependent prolyl-hydroxylases. Hypoxia also affects virulence and antibiotic resistance in bacterial pathogens. However, less is known about the impact of hypoxia on host-pathogen interactions such as bacterial adhesion and infection. In the current study, we demonstrate that hypoxia decreases the internalization of P. aeruginosa into cultured epithelial cells resulting in decreased host cell death. This response can also be elicited by the hydroxylase inhibitor Dimethyloxallyl Glycine (DMOG. Reducing HIF-2α expression or Rho kinase activity diminished the effects of hypoxia on P. aeruginosa infection. Furthermore, in an in vivo pneumonia infection model, application of DMOG 48 h before infection with P. aeruginosa significantly reduced mortality. Thus, hypoxia reduces P. aeruginosa internalization into epithelial cells and pharmacologic manipulation of the host pathways involved may represent new therapeutic targets in the treatment of P. aeruginosa infection.

  16. Aquaporin 2 promotes cell migration and epithelial morphogenesis.

    Science.gov (United States)

    Chen, Ying; Rice, William; Gu, Zhizhan; Li, Jian; Huang, Jianmin; Brenner, Michael B; Van Hoek, Alfred; Xiong, Jianping; Gundersen, Gregg G; Norman, Jim C; Hsu, Victor W; Fenton, Robert A; Brown, Dennis; Lu, Hua A Jenny

    2012-09-01

    The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin β1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin β1 by mutating the RGD motif led to reduced endocytosis, retention of integrin β1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin β1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.

  17. Changes in central corneal thickness and endothelial cell count pediatric cataract surgery

    International Nuclear Information System (INIS)

    Memon, M.N.

    2015-01-01

    To evaluate the mean changes in Central Corneal Thickness (CCT) and Endothelial Cell Count (ECC) in eyes after pediatric cataract surgery with foldable intraocular lens using scleral tunnel incision micro-surgical technique. Study Design: Qausi experimental study. Place and Duration of Study: Department of Pediatric Ophthalmology and Strabismus, Al-Shifa Trust Eye Hospital, Rawalpindi, from May 2011 to March 2012. Methodology: Fifty-two eyes of 37 children with pediatric cataract were included in the study. Extracapsular Cataract Extraction (ECE) with foldable Intra Ocular Lens (IOL) implantation using sclera tunnel incision was performed in all children. Endothelial Cell Count (ECC) and Central Corneal Thickness (CCT) were recorded before surgery and 1 month, 3 months and 6 months after surgery and the effect of currently practiced surgical technique on ECC and CCTwas evaluated. Results: The mean age at the time of surgery was 8.8 ± 2.7 years (range: 4 to 15 years). The postoperative ECC and CCT were significantly different from the pre-operative values. Mean pre-operative ECC was 3175.3 ± 218.4 cell/mm2 and in first postoperative month the mean ECC was 3113.4 ± 210.8 cell/mm2 (p<0.0001). In the 3rd and 6th month postoperative means ECC were 3052 ± 202.5 cell/mm2 (p<0.0001) and 3015 ±190.6 cell/mm2 (p<0.0001), respectively. The mean cell loss at first postoperative month was 1.95% and at 3rd and 6th postoperative month were 3.9% and 5.05%, respectively. Mean pre-operative CCT was 514 ± 49.9 micro m and first postoperative mean CCT after 1 month was 524.1 ± 25 micro m (p = 0.084). After the 3rd and 6th months postoperative, mean CCT were 527.3 ± 24.6 micro m, and 530 ± 24.5 micro m, respectively. Third and 6th months postoperative means were significantly higher than baseline CCT, p = 0.024 and 0.007, respectively. Conclusion: Endothelial cell loss with closed chamber micro-surgical technique using scleral tunnel incision is within acceptable limits and

  18. Renal response assayed by survival of tubule epithelial cells

    International Nuclear Information System (INIS)

    Withers, H.R.; Mason, K.A.

    1985-01-01

    The epithelium of the renal tubules is essentially non-proliferative and hence is slow to be depleted after irradiation. Ultimately, however, depletion occurs. If cells survive within a tubule they regenerate the epithelial lining. After higher doses, e.g. greater than 12 Gy, some tubules are completely depopulated of epithelium giving rise to a histological picture of empty tubules interspersed with regenerated tubules. It is assumed that nephrons are all essentially the same size, that cell survival is a random probability and that, therefore, when a proportion of tubules are completely devoid of epithelium, those that aren't have regenerated from one or a few cells, the distribution of numbers of survivors per tubule following Poisson statistics. Based on these assumptions it is possible to determine a dose-survival relationship for renal tubule cells

  19. Expression of ICAM-1 in colon epithelial cells

    DEFF Research Database (Denmark)

    Vainer, Ben; Sørensen, Susanne; Seidelin, Jakob

    2003-01-01

    on monolayers of cancer cells. Conflicting results exist on epithelial ICAM-1 expression, and the aim of this study was to compare the expression in various models of colonic epithelium. MATERIALS AND METHODS: Colonic biopsies from four UC patients and four controls were examined by cryoimmuno......-electron microscopy using ICAM-1-antibodies. In four other controls, the epithelium was isolated from colonic biopsies, embedded in collagen, and evaluated similarly. Isolated crypts and cultured cancer cells were stimulated with interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha). RESULTS: ICAM-1......, both colonocytes and HT29 cells were capable of expressing ICAM-1 on their apical membranes in response to supraphysiologic cytokine concentrations. These observations question the justification of extrapolating observations from colon cancer cell lines to in vivo inflammatory conditions....

  20. MAST CELLS AND ANGIOGENESIS IN ORAL EPITHELIAL DYSPLASTIC LESIONS AND ORAL SQUAMOUS CELL CARCINOMA

    Directory of Open Access Journals (Sweden)

    Veda, Marla Vinay

    2015-01-01

    Full Text Available Background: The progression of oral epithelial dysplastic lesions into oral squamous cell carcinoma is characterized by an ‘angiogenic switch’ which is characterized by an increase in neo-vascularization in the sub-epithelial lamina propria which can be considered an indicator of malignant transformation. Mast cells are a rich source of various angiogenic factors. Moreover mast cells secrete various proteolytic enzymes which degrade the extracellular matrix and create space for the developing blood vessels. Aims: This study was undertaken to determine the relationship between mast cell density and microvessel density in normal oral mucosa, oral epithelial dysplasia and oral squamous cell carcinoma and to find out whether any correlation exists between these two parameters. Material and Methods: This retrospective study was performed using formalin fixed, paraffin embedded tissues of previously diagnosed cases of oral epithelial dysplasia and oral squamous cell carcinoma. Mast cells were stained using toluidine blue, whereas in the capillaries, immunohistochemical staining technique was performed using mouse monoclonal antibody against CD34. Results: Mast cell density and microvessel density were higher in oral epithelial dysplasia and in oral squamous cell carcinoma compared to the normal mucosa. However, statistically significant positive correlation was noted only in oral epithelial dysplasia Conclusion: The above results probably indicate a role of mast cells in ‘angiogenic switch’. These angiogenic factors secreted by mast cells promote angiogenesis either directly by stimulating the migration and/or proliferation of mast cells or indirectly through degradation of extracellular matrix. Targeting the mast cells may contribute in preventing the progression of the lesion.

  1. Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells

    International Nuclear Information System (INIS)

    Roberts, Joan E.; Wielgus, Albert R.; Boyes, William K.; Andley, Usha; Chignell, Colin F.

    2008-01-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C 60 (OH) 22-26 ] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C 60 (OH) 22-26 in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 μM. Exposure to either UVA or visible light in the presence of > 5 μM fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 μM lutein, 1 mM N-acetyl cysteine, or 1 mM L-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA > visible light > dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein α-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C 60 (OH) 22-26 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo

  2. In vivo analysis of stromal integration of multilayer amniotic membrane transplantation in corneal ulcers.

    Science.gov (United States)

    Nubile, Mario; Dua, Harminder S; Lanzini, Manuela; Ciancaglini, Marco; Calienno, Roberta; Said, Dalia G; Pocobelli, Augusto; Mastropasqua, Rodolfo; Carpineto, Paolo

    2011-05-01

    To evaluate integration of amniotic membrane into the corneal stroma using laser scanning in vivo confocal microscopy and anterior segment optical coherence tomography (AS-OCT). Prospective noncomparative interventional case series. Twenty-two eyes of 22 consecutive patients (mean age 53.9 ± 9.2 years) presenting with noninfectious corneal ulcers and stromal thinning unresponsive to medical treatment were enrolled. Multiple layers of amniotic membrane were applied over the ulcer bed to fill the ulcer crater and held in place with an overlying amniotic membrane patch, which was anchored to the surrounding cornea with 10-0 nylon interrupted sutures. Outcome measures were healing of the corneal ulcers, corneal morphology and stromal thickness changes at the ulcer site as measured by AS-OCT and surface epithelialization, stromal repopulation, and structural modifications of the amniotic membrane grafts as evaluated by confocal microscopy. Follow-up extended to 12 months. Successful result was observed in 20 of 22 eyes (90.9%). AS-OCT showed that the mean residual stromal thickness at the ulcer bed was 222 ± 70 μm before surgery. The mean thickness of amniotic membrane layers at the same site was 394 ± 80 μm while the mean total corneal thickness was 623 ± 51 μm at day 1 post surgery. Thereafter a progressive reduction in thickness to 420 ± 61 μm at 6 months occurred, after which the thickness stabilized. Confocal microscopy showed that integration of the amniotic membrane tissues with corneal stroma was preceded by epithelialization over the amniotic membrane covering the ulcer. This occurred 15 ± 5 days post surgery in the successful cases. Confocal microscopy also showed that the amniotic membrane patch was degraded during the first few weeks after surgery, while the integrated amniotic tissues underwent progressive modifications characterized by early loss of amniotic epithelial cells, changes in fibrillar structure, and migration into the amniotic stroma

  3. Aspects of cell proliferation in oral epithelial dysplastic lesions.

    Science.gov (United States)

    Oliver, R J; MacDonald, D G; Felix, D H

    2000-02-01

    There is a need for objective methods of assessment of oral epithelial precancerous lesions and reliable markers for the prediction of malignant change in these lesions. Cell proliferation was examined in 20 dysplastic lesions from the tongue and floor of mouth using bromodeoxyuridine (BrdU) and Ki-67, and a histological compartment analysis was performed. Half of a fresh biopsy from each case was incubated in BrdU for 15 min, the other half was routinely processed and used for Ki-67 analysis. Sections from each block were immunohisto chemically stained with antibodies against BrdU and Ki-67. Dysplasia was graded according to the method of Smith & Pindborg. The BrdU labelling index (LI) and the growth fraction (GF), assessed by the use of Ki-67, was quantified and expressed as units per millimetre basement membrane length (BL) and per 100 total nucleated cells (TNC). The mean LI/TNC was 10.87 (SD 3.65) and the mean LI/BL was 51.55 (SD 20.75). The mean GF/TNC was 26.66 (SD 17.78) and GF/BL was 157.07 (SD 125.84). The mean epithelial thickness was 229.09 microm (SD 104.73). The LI/BL correlated with the atypia score and with the GF/BL. The progenitor compartment sizes also correlated with the atypia scores. The BrdU labelling index provides a further objective measurement of oral epithelial dysplasia and the progenitor compartments were large, implying that basal cell hyperplasia is a significant component of the dysplasia.