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Human Corneal Epithelial Cells (human + corneal_epithelial_cell)

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Medical Sciences	90%

Selected Abstracts

4363: Cytostatic and cytotoxic effects of 5-fluorouracil on human corneal epithelial cells and keratocytes

ACTA OPHTHALMOLOGICA, Issue 2010
E MIDENA
Purpose To investigate the effects of different 5-Fluorouracil (5-FU) concentrations, exposure times and application techniques on in vitro cultured human corneal cells. Methods Human corneal epithelial cells (HCEC) and human corneal keratocytes (HCK) cultures were exposed to different 5-FU concentration (0.025% to 1%) and incubation times (single 5' to 2 hrs). The cytostatic effect was evaluated as ratio of inhibition of migration towards controls. The cytotoxic effect evaluation included both contrast phase microscopic observations, and viability measures performed using a MTT[3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide; thiazolyl blue] colorimetric assay. The results were expressed as ratio of optical density (OD) reduction 24 hrs after exposures. Results Cytostatic effect is time and dose dependent. Minimal inhibiting dose (ID50) was 0,55% after 1 hour incubation for HCEC; ID50 was 0,5% after 2 hrs incubation for HCK . A 100% inhibitory effect was never observed at any concentration or incubation interval. No cytotoxic changes were observed at 5-FU < 1%; 5-FU 1% showed time-dependent cytotoxic changes just in HCEC cultures. MTT analysis showed no OD reduction at 5-FU concentration < 1%, whereas 1% 5-FU showed OD reduction < 50% at any tested exposure time. HCEC showed higher reduction in OD than HCK. Conclusion 5-FU shows no definite signs of irreversible toxicity to normal cultured corneal epithelial and keratocyte cells at examined concentrations and exposure time. [source]

Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells

IMMUNOLOGY, Issue 1 2006
Ashok Kumar
Summary The objective of this study was to examine the expression of Toll-like receptor 3 (TLR3) by human corneal epithelial cells (HCECs) and to determine whether exposure to the TLR3 agonist polyinosinic-polycytidylic acid [poly(I:C)]induces an antiviral response in these cells. Fluorescence-activated cell sorter (FACS) analysis revealed TLR3 to be constitutively expressed and distributed intracellularly in HCECs. Stimulation of HCECs with the TLR3 agonist poly(I:C) induced the activation of nuclear factor (NF)-,B and production of the proinflammatory cytokine interleukin (IL)-6 and the chemokine IL-8. Upon exposure to poly(I:C), HCECs initiated a potent antiviral response resulting in an increase of interferon (IFN)-, mRNA expression (7-fold). Poly(I:C) stimulation also up-regulated mRNA expression of the antiviral chemokine IFN-, inducible protein 10 (IP10), myxovirus resistance gene A and 2,,5,-oligoadenylate synthetase (5-, 10- and 9-fold, respectively), and secretion of IP10. These responses were also induced by exogenously added type 1 IFNs, but could not be blocked by pretreatment of the cells with anti-TLR3 monoclonal antibody, suggesting that the receptor was not expressed on the cell surface. Furthermore, incubation of HCECs with an endosomal acidification inhibitor, chloroquine, markedly inhibited poly(I:C)-mediated IFN-, expression in HCECs. These results suggest that corneal epithelial cells are important sentinels of the corneal innate immune system against viral infection, and that stimulation of TLR3 can induce the expression of key proinflammatory cytokines and chemokines and antiviral genes that help in the defence of the cornea against viral infection. [source]

Effluxing ABC transporters in human corneal epithelium

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2010
Kati-Sisko Vellonen
Abstract ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1,6 (MRP1,6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1087,1098, 2010 [source]

4363: Cytostatic and cytotoxic effects of 5-fluorouracil on human corneal epithelial cells and keratocytes

Characterization of efflux proteins in human corneal epithelial cells

ACTA OPHTHALMOLOGICA, Issue 2007
KS VELLONEN
Purpose: Corneal epithelium is the main barrier for absorption of drugs into intraocular tissues after topical administration and part of this barrier may be formed by efflux proteins which translocate molecules from the cell interior to the extracellular space. The aim of this study was to characterize the gene expression and the activity of the efflux transporters in the cell culture model of immortalized human corneal epithelial cells (HCE cells), in primary cell line (HCEpiC), and in the human corneal epithelium. Methods: The mRNA levels of MDR1, MRP1-MRP6, and BCRP were determined by the quantitative RT-PCR. Immunohistochemistry was used to study protein expression and localization of efflux transporters. Functionality of these proteins was assessed with calcein-AM efflux assay and by measuring the efflux of CDCF. Furthermore, bidirectional permeability of rhodamine 123 (Rh123) was studied. Results: The mRNA of MRP1 and MRP5 were detected in the human cornea and in both cell lines. These efflux proteins were found in the cell membranes of the human corneal epithelium. At mRNA level some efflux proteins were over-expressed in the HCE and the primary cell lines. Increased calcein retention and decreased CDCF efflux in the presence of inhibitors suggested efflux protein activity in both primary and HCE cells. Likewise, directionality in Rh123 permeability was diminished in the presence of verapamil in HCE model. Conclusions: Functionality of the efflux proteins was demonstrated in the human corneal epithelial cells. MRP1 and MRP5 proteins may have important protecting role in corneal surface by transporting molecules out from the epithelial cells. It seems that the efflux activity in the HCE model differs from that of the corneal epithelium in vivo [source]

4,-PDD induces Ca2+ influx in human corneal epithelial cells by activating TRPV4 channels

ACTA OPHTHALMOLOGICA, Issue 2007
S MERGLER
Purpose: Transient receptor potential (TRP) isoform expression is evident in human corneal epithelial cells (HCEC-SV40). However, their role in maintaining corneal epithelial homeostasis is not fully understood. We probed for gene and protein expression as well as functional activity of the vanilloid subtype, TRPV4, in immortalized HCEC-SV40 since they elicit Ca2+ dependent regulatory volume decrease (RVD) responses during exposure to a hypotonic challenge. Methods: RT-PCR and Western blotting analyses identified TRPV4 gene and protein expression. Functional activity was assessed based on determining whether the TRPV4 selective agonist, 4,-PDD, induced transients increases in intracellular Ca2+ concentration. Results: Single cell fluorescence imaging results showed that 4,-PDD (3 ,M) increased intracellular Ca2+ concentration. The fura2 fluorescence ratio (f340/f380) was 0.39 ± 0.03578 in the resting state (n = 5). After application of 4,-PDD it increased to 0.904 ± 0.14363 (n = 5; p = 5.72077×10-5). This increase was abolished by the TRP channel blocker ruthenium red or by Ca2+-free Ringer's medium. Conclusions: In conclusion, there is functional TRPV4 expression in HCEC-SV40. TRPV4 expression may provide an osmosensor role to induce RVD behavior during exposure to a hypotonic challenge since this response is mediated through intracellular Ca2+ transients. Supported in part DFG Pl 150/14-1 and NIH, EY04795. CR: none [source]

Longterm survival of transplanted human corneal epithelial cells and corneal stem cells

ACTA OPHTHALMOLOGICA, Issue 4 2005
Maria Egarth
Abstract. Purpose:,To investigate the survival of donor-derived epithelial cells in conventional penetrating keratoplasty (PKP) and in homologous penetrating central limbal keratoplasty (HPCLK). Methods and Patients:,Epithelial cells from 26 eyes of 26 patients were analysed. All cases were sex-mismatched (i.e. the transplant and patient were of different genders). At suture removal more than 1 year post surgery, epithelial cells were obtained by gently wiping the removed sutures on glass slides. The cell samples were analysed using fluorescent in situ hybridization (FISH) of the sex chromosomes. This technique makes it possible to allocate the origin of each cell nucleus to either the donor or the recipient. Results:,All 19 conventional PKPs were clear and seven had donor-derived epithelial cells at suture removal. Five of the seven HPCLK grafts were clear at the time of investigation (365,1355 days post surgery), and donor-derived epithelial cells were found in two grafts. Conclusion:,Harvesting cells from removed sutures in combination with FISH enables the clinical study of cell survival in corneal transplants without jeopardizing functioning grafts. From the limited sample investigated, the following tentative conclusions can be made. Donor-derived epithelial cells can remain in conventional PKP for over 1 year. In combined stem cell and corneal grafts (HPCLK), donor-derived epithelial cells may also be retrieved at 1 year or beyond following surgery but the correlation between their presence and a remaining clear graft is uncertain. [source]

Serotype and adhesion of Pseudomonas aeruginosa isolated from contact lens wearers

CLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 3 2001
Sophy J Thuruthyil PhD
ABSTRACT The purpose of the present study was to correlate the serotypes of Pseudomonas aeruginosa to the bacterial adhesion to contact lenses and human corneal epithelial cells. Twenty-three strains isolated from contact lens wearers were used for the study. The bacterial serotypes were examined with a P. aeruginosa antisera kit. The attachment of bacteria on contact lenses or human corneal epithelial cells was determined by counting the number of adhered bacteria after incubation of the bacteria with contact lenses or corneal epithelial cells. The 23 ocular isolates belonged to seven serotypes. Strains of serotypes I, G and E were the three dominant serogroups and were more adhesive to contact lenses compared with other groups of the bacteria. The bacterial serotypes and the clinical sequelae were not strongly related. These results indicate that the surface characteristics of bacterial serotypes are related to the bacterial adhesion to the surface, but the pathogenesis of the bacteria may result from multiple factors. [source]