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Sample records for human corneal epithelium

  1. Effluxing ABC Transporters in Human Corneal Epithelium

    PubMed Central

    Vellonen, Kati-Sisko; Mannermaa, Eliisa; Turner, Helen; Häkli, Marika; Wolosin, J. Mario; Tervo, Timo; Honkakoski, Paavo; Urtti, Arto

    2010-01-01

    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. PMID:19623615

  2. Chloride Channels and Transporters in Human Corneal Epithelium

    PubMed Central

    Cao, Lin; Zhang, Xiao-Dong; Liu, Xiaobo; Chen, Tsung-Yu; Zhao, Min

    2010-01-01

    Transport of water and electrolytes is critical for corneal clarity. Recent studies indicate another important function of transport of ions and electrolytes - establishing wound electric fields that guide cell migration. We found chloride (Cl-) flux is a major component of the corneal wound electric current. In order to elucidate the mechanisms of Cl- transport, we studied Cl- channels and transporters in human corneal epithelial (HCE) cells. We tested a transformed human corneal epithelial cell line (tHCE), primary cultures of human corneal epithelial cells (pHCE), and human donor corneas. We first used RT-PCR to determine expression levels of mRNA of CLC (Cl- channel/transporter) family members and CFTR (cystic fibrosis transmembrane conductance regulator) in HCE cells. We then confirmed protein expression and distribution of selected CLC family members and CFTR with Western blot and immuno-fluorescence confocal microscopy. Finally, Cl- currents were recorded with electrophysiological techniques. The mRNAs of CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, and CFTR were detected in the HCE cell line. CLC-1 and CLC-7 were not detectable. Western blot and immunostaining confirmed protein expression and distribution of CLC-2, CLC-3, CLC-4, CLC-6 and CFTR in human corneal epithelium. CLC-2 preferentially labeled the apical and basal layers, while CLC-3 and CLC-4 labeled only the superficial layer. CLC-6 and CFTR labeling showed a unique gradient with strong staining in apical layers which gradually decreased towards the basal layers. Corneal endothelium was positive for CLC-2, CLC-3, CLC-4, CLC-6 and possibly CFTR. Human corneal epithelial cells demonstrated voltage dependent Cl- currents. HCE cells express functional Cl- channels and transporters. CLC-2, CLC-3, CLC-4, CLC-6, and CFTR had distinct expression patterns in human corneal epithelium. Those molecules and their distribution may play important roles in maintaining resting Cl- fluxes and in regulating Cl- flux at corneal

  3. Development of human corneal epithelium on organized fibrillated transparent collagen matrices synthesized at high concentration.

    PubMed

    Tidu, Aurélien; Ghoubay-Benallaoua, Djida; Lynch, Barbara; Haye, Bernard; Illoul, Corinne; Allain, Jean-Marc; Borderie, Vincent M; Mosser, Gervaise

    2015-08-01

    Several diseases can lead to opacification of cornea requiring transplantation of donor tissue to restore vision. In this context, transparent collagen I fibrillated matrices have been synthesized at 15, 30, 60 and 90 mg/mL. The matrices were evaluated for fibril organizations, transparency, mechanical properties and ability to support corneal epithelial cell culture. The best results were obtained with 90 mg/mL scaffolds. At this concentration, the fibril organization presented some similarities to that found in corneal stroma. Matrices had a mean Young's modulus of 570 kPa and acellular scaffolds had a transparency of 87% in the 380-780 nm wavelength range. Human corneal epithelial cells successfully colonized the surface of the scaffolds and generated an epithelium with characteristics of corneal epithelial cells (i.e. expression of cytokeratin 3 and presence of desmosomes) and maintenance of stemness during culture (i.e. expression of ΔNp63α and formation of holoclones in colony formation assay). Presence of cultured epithelium on the matrices was associated with increased transparency (89%).

  4. Automated image classification applied to reconstituted human corneal epithelium for the early detection of toxic damage

    NASA Astrophysics Data System (ADS)

    Crosta, Giovanni Franco; Urani, Chiara; De Servi, Barbara; Meloni, Marisa

    2010-02-01

    For a long time acute eye irritation has been assessed by means of the DRAIZE rabbit test, the limitations of which are known. Alternative tests based on in vitro models have been proposed. This work focuses on the "reconstituted human corneal epithelium" (R-HCE), which resembles the corneal epithelium of the human eye by thickness, morphology and marker expression. Testing a substance on R-HCE involves a variety of methods. Herewith quantitative morphological analysis is applied to optical microscope images of R-HCE cross sections resulting from exposure to benzalkonium chloride (BAK). The short term objectives and the first results are the analysis and classification of said images. Automated analysis relies on feature extraction by the spectrum-enhancement algorithm, which is made sensitive to anisotropic morphology, and classification based on principal components analysis. The winning strategy has been the separate analysis of the apical and basal layers, which carry morphological information of different types. R-HCE specimens have been ranked by gross damage. The onset of early damage has been detected and an R-HCE specimen exposed to a low BAK dose has been singled out from the negative and positive control. These results provide a proof of principle for the automated classification of the specimens of interest on a purely morphological basis by means of the spectrum enhancement algorithm.

  5. Expression of vitamin D receptor and cathelicidin in human corneal epithelium cells during fusarium solani infection

    PubMed Central

    Cong, Lin; Xia, Yi-Ping; Zhao, Gui-Qiu; Lin, Jing; Xu, Qiang; Hu, Li-Ting; Qu, Jian-Qiu; Peng, Xu-Dong

    2015-01-01

    AIM To observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide (CAMP) in the downstream pathway of VDR. METHODS Immunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction (qPCR) was performed to observe the messenger ribonucleic acid (mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both mRNA and protein levels. RESULTS We found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The mRNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen (P<0.01) and it could be inhibited by toll like receptor 2 (TLR2) monoclonal antibody. The CAMP expression was decreased because of fusarium solani antigen stimulation (P<0.01). CONCLUSION The VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen. PMID:26558193

  6. Expression of vitamin D receptor and cathelicidin in human corneal epithelium cells during fusarium solani infection.

    PubMed

    Cong, Lin; Xia, Yi-Ping; Zhao, Gui-Qiu; Lin, Jing; Xu, Qiang; Hu, Li-Ting; Qu, Jian-Qiu; Peng, Xu-Dong

    2015-01-01

    To observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide (CAMP) in the downstream pathway of VDR. Immunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction (qPCR) was performed to observe the messenger ribonucleic acid (mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both mRNA and protein levels. We found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The mRNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen (P<0.01) and it could be inhibited by toll like receptor 2 (TLR2) monoclonal antibody. The CAMP expression was decreased because of fusarium solani antigen stimulation (P<0.01). The VDR expression can be increased via TLR2/1-VDR pathway while the CAMP expression is decreased by the stimulation of fusarium solani antigen.

  7. Impact of the environment on the mammalian corneal epithelium.

    PubMed

    Ringvold, Amund; Anderssen, Erlend; Kjønniksen, Inge

    2003-01-01

    To evaluate whether the content of ascorbic acid in the corneal epithelium and aqueous humor reflects seasonal fluctuations in parallel with environmental changes. Reindeer, cattle, rabbits, and humans were examined, to cover a broad spectrum of overlapping habitats. Ascorbic acid was determined by high-performance liquid chromatography. The thickness of the corneal epithelium was measured, and the number of cells was counted in the tissue sections. Three groups of reindeer eyes were used, two of them collected during summer, the third group during winter. Ascorbate content did not show seasonal variation in either the corneal epithelium or the aqueous humor, whereas epithelial thickness and number of cells decreased significantly from summer to winter. In cattle, ascorbate content, thickness of the epithelium, and number of cells were lower in animals tended indoors compared with those tended outdoors, whereas ascorbate level in the aqueous humor remained similar in both cases. The rabbit showed significantly reduced ascorbate content in the corneal epithelium but not in the aqueous humor in tarsorrhaphy-treated eyes. This procedure did not change epithelial thickness, but the number of cells was slightly increased. The mean epithelial thickness in human corneas successively decreased with increasing latitude and decreasing radiation exposure from the summer season in Oslo to the midnight sun, polar night, conditions in Tromsø, 10 degrees far north, although the differences did not reach statistical significance. Ambient radiation is needed to sustain high ascorbic acid concentration in the corneal epithelium. Corneal epithelial thickness and number of cells are prone to seasonal fluctuations regulated by ambient radiation. In contrast, ascorbate content of the aqueous humor is uninfluenced by environmental change. It is suggested that seasonal adaptation of mammalian corneal epithelium in response to variation in ambient radiation may be nature's strategy for

  8. OVOL2 Maintains the Transcriptional Program of Human Corneal Epithelium by Suppressing Epithelial-to-Mesenchymal Transition.

    PubMed

    Kitazawa, Koji; Hikichi, Takafusa; Nakamura, Takahiro; Mitsunaga, Kanae; Tanaka, Azusa; Nakamura, Masahiro; Yamakawa, Tatsuya; Furukawa, Shiori; Takasaka, Mieko; Goshima, Naoki; Watanabe, Akira; Okita, Keisuke; Kawasaki, Satoshi; Ueno, Morio; Kinoshita, Shigeru; Masui, Shinji

    2016-05-10

    In development, embryonic ectoderm differentiates into neuroectoderm and surface ectoderm using poorly understood mechanisms. Here, we show that the transcription factor OVOL2 maintains the transcriptional program of human corneal epithelium cells (CECs), a derivative of the surface ectoderm, and that OVOL2 may regulate the differential transcriptional programs of the two lineages. A functional screen identified OVOL2 as a repressor of mesenchymal genes to maintain CECs. Transduction of OVOL2 with several other transcription factors induced the transcriptional program of CECs in fibroblasts. Moreover, neuroectoderm derivatives were found to express mesenchymal genes, and OVOL2 alone could induce the transcriptional program of CECs in neural progenitors by repressing these genes while activating epithelial genes. Our data suggest that the difference between the transcriptional programs of some neuroectoderm- and surface ectoderm-derivative cells may be regulated in part by a reciprocally repressive mechanism between epithelial and mesenchymal genes, as seen in epithelial-to-mesenchymal transition.

  9. The oxidant role of 4-hydroxynonenal in corneal epithelium.

    PubMed

    Chen, Longlong; Zong, Rongrong; Zhou, Jing; Ge, Lianping; Zhou, Tong; Ma, Jian-xing; Liu, Zuguo; Zhou, Yueping

    2015-05-29

    4-Hydroxynonenal (4-HNE or HNE) is a main endogenous product of cellular lipid peroxidation in tissues and is reported to play pathogenic roles in eye diseases. Here we investigated the association between 4-HNE and oxidative stress in the corneal epithelium. 4-HNE suppressed the cell viability of human corneal epithelial cells (HCE) in a concentration dependent manner. 4-HNE significantly increased the level of 3-Nitrotyrosine (3-NT), a marker of oxidative stress, in HCE cells and corneal epithelium of rats by immunofluorescent staining and Western blot analysis. To its underlying mechanistic on ROS system, 4-HNE elevated the ROS generation enzyme NADPH oxidase 4 (NOX4) and induced the activation of NF-E2-related factor-2 (NRF2) and its downstream effectors: NAD(P)H dehydrogenase (quinone 1) (NQO1) and glutathione S-transferase P (GSTP). Furthermore, N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the inhibitory and oxidant effects of 4-HNE on the corneal epithelial cells. In conclusion, 4-HNE plays an oxidant role in the corneal epithelium and this work provides a new strategy for the pathogenesis and treatment of corneal diseases.

  10. A Novel Innate Response of Human Corneal Epithelium to Heat-killed Candida albicans by Producing Peptidoglycan Recognition Proteins

    PubMed Central

    Hua, Xia; Yuan, Xiaoyong; Li, Zhijie; Coursey, Terry G.; Pflugfelder, Stephen C.; Li, De-Quan

    2015-01-01

    Fungal infections of the cornea can be sight-threatening and have a worse prognosis than other types of microbial corneal infections. Peptidoglycan recognition proteins (PGLYRP), which are expressed on the ocular surface, play an important role in the immune response against bacterial corneal infections by activating toll-like receptors (TLRs) or increasing phagocytosis. However, the role of PGLYRPs in innate immune response to fungal pathogens has not been investigated. In this study, we observed a significant induction of three PGLYRPs 2–4 in primary human corneal epithelial cells (HCECs) exposed to live or heat-killed Candida albicans (HKCA). The C-type lectin receptor dectin-1 plays a critical role in controlling Candida albicans infections by promoting phagocytic activity and cytokine production in macrophages and dendritic cells. Here, we demonstrate that dectin-1 is expressed by normal human corneal tissue and primary HCECs. HKCA exposure increased expression of dectin-1 on HCECs at mRNA and protein levels. Interestingly, dectin-1 neutralizing antibody, IκB-α inhibitor BAY11-7082, and NF-κB activation inhibitor quinazoline blocked NF-κB p65 nuclear translocation, as well as the induction of the PGLYRPs by HKCA in HCECs. Furthermore, rhPGLYRP-2 was found to suppress colony-forming units of Candida albicans in vitro. In conclusion, these findings demonstrate that dectin-1 is expressed by human corneal epithelial cells, and dectin-1/NF-κB signaling pathway plays an important role in regulating Candida albicans/HKCA-induced PGLYRP secretion by HCECs. PMID:26039076

  11. Expression of JAM-A in the Human Corneal Endothelium and Retinal Pigment Epithelium: Localization and Evidence for Role in Barrier Function

    PubMed Central

    Mandell, Kenneth J.; Berglin, Lennart; Severson, Eric A.; Edelhauser, Henry F.; Parkos, Charles A.

    2007-01-01

    Purpose Junctional adhesion molecules (JAMs) are a family of adhesion proteins found in intercellular junctions. Evidence suggests that JAM-A is important for the regulation of tight junction assembly and epithelial barrier function. The authors recently reported that JAM-A is expressed in rabbit corneal endothelium and that antibody to JAM-A produces corneal swelling. In the present study, they investigate JAM-A expression in the human corneal endothelium and retinal pigment epithelium (RPE) and examine the effect of a function-blocking antibody to JAM-A on the permeability of cultured RPE cell monolayers. Methods Expression of JAM-A in human corneal endothelium, human RPE tissue, and cultured ARPE-19 monolayers was assessed by immunofluorescence confocal microscopy. Localization of JAM-A was compared with the tight junction-associated protein zonula occludens-1 (ZO-1). To investigate JAM-A function in ARPE-19 cells, ARPE-19 monolayers were subjected to a calcium switch protocol to disrupt cell junctions and treated with a function-blocking antibody to JAM-A or an isotype-matched control. Dextran flux assays were performed to assess the effect of JAM-A antibody on ARPE-19 monolayer permeability. Results Expression of JAM-A was observed in human corneal endothelium, and its distribution correlated with the tight junction-associated protein ZO-1. In addition, expression of JAM-A was observed in human RPE and in intercellular junctions of ARPE-19 monolayers. The localization pattern of JAM-A in the RPE and ARPE-19 monolayers was similar to that of ZO-1. ARPE-19 monolayers treated with antibody to JAM-A demonstrated a 33% increase in permeability to 10,000 MWt dextran compared with monolayers treated with control antibody. Conclusions Results of this study provide new information about JAM-A expression in tight junctions of the human corneal endothelium and human RPE. The observation that antibodies to JAM-A increase ARPE-19 monolayer permeability is consistent with

  12. Molecular Evidence and Functional Expression of a Novel Drug Efflux pump (ABCC2) in Human Corneal Epithelium and Rabbit Cornea and its role in Ocular drug efflux

    PubMed Central

    Karla, Pradeep K.; Pal, Dhananjay; Quinn, Tim; Mitra, Ashim K.

    2007-01-01

    Cornea is considered as a major barrier for ocular drug delivery. Low ocular bioavailability of drugs has been attributed primarily to low permeability across corneal epithelium thus leading to sub-therapeutic concentrations of drug in the eye and treatment failure. The role of drug efflux proteins, particularly the Pglycoprotein in ocular drug bioavailability has been reported. The objective of this research was to determine whether human corneal epithelium expresses multi drug resistance associated proteins contributing to drug efflux by employing both cultured corneal cells and freshly excised rabbit cornea. SV40 HCEC and rPCEC were selected for in-vitro testing. SV40-HCEC and freshly excised rabbit corneas were utilized for transport studies. [3H]-cyclosporine-A and [14C]-erythromycin which are known substrates for ABCC2 and MK-571, a specific inhibitor for MRP were applied in this study. RT-PCR indicated a unique and distinct band at ∼272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band ∼181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at ∼190-kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [3H]-cyclosporine-A and [14C]-erythromycin in the presence of MK-571 was significantly enhanced than control in both SV40 HCEC and rPCEC. Similarly a significant elevation in (A→B) permeability of [3H]-cyclosporine-A and [14C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A→B transport of [3H]-cyclosporine-A was elevated in the presence of MK-571 in freshly excised rabbit cornea indicating potential role of this efflux transporter and high clinical significance of this finding. PMID:17156953

  13. A new 3D reconstituted human corneal epithelium model as an alternative method for the eye irritation test.

    PubMed

    Jung, Kyoung-Mi; Lee, Su-Hyon; Ryu, Yang-Hwan; Jang, Won-Hee; Jung, Haeng-Sun; Han, Ju-Hee; Seok, Seung-Hyeok; Park, Jae-Hak; Son, Youngsook; Park, Young-Ho; Lim, Kyung-Min

    2011-02-01

    Many efforts are being made to develop new alternative in vitro test methods for the eye irritation test. Here we report a new reconstructed human corneal epithelial model (MCTT HCE model) prepared from primary-cultured human limbal epithelial cells as a new alternative in vitro eye irritation test method. In histological and immunohistochemical observation, MCTT HCE model displayed a morphology and biomarker expressions similar to intact human cornea. Moreover, the barrier function was well preserved as measured by high transepithelial electrical resistance, effective time-50 for Triton X-100, and corneal thickness. To employ the model as a new alternative method for eye irritation test, protocol refinement was performed and optimum assay condition was determined including treatment time, treatment volume, post-incubation time and rinsing method. Using the refined protocol, 25 reference chemicals with known eye irritation potentials were tested. With the viability cut-off value at 50%, chemicals were classified to irritant or non-irritant. When compared with GHS classification, the MCTT HCE model showed the accuracy of 88%, sensitivity of 100% and specificity of 77%. These results suggest that the MCTT HCE model might be useful as a new alternative eye irritation test method. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. Cytokeratin patterns in corneal, limbal, and conjunctival epithelium. An immunofluorescence study with PKK-1, 8.12, 8.60, and 4.62 anticytokeratin antibodies.

    PubMed

    Ryder, M I; Weinreb, R N

    1990-11-01

    The authors examined immunofluorescently the specific cytokeratin staining patterns of corneal, limbal, and conjunctival epithelium with PKK-1, 8.12, 4.62, and 8.60 monoclonal anticytokeratin antibodies. Observations were made on unfixed frozen postmortem human tissue. The PKK-1 antibody stain was observed in all layers of corneal epithelium but only in suprabasal layers of limbal and conjunctival epithelium. By contrast, the 8.12 antibody stain was observed only in the superficial layer of corneal epithelium but through all layers of limbal and conjunctival epithelium. The 4.60 antibody stain was seen in focal areas of corneal and limbal epithelium and through all layers of conjunctival epithelium. The 8.60 antibody stain was not present in the three epithelia. These immunofluorescence studies showed unique cytokeratin patterns among layers in corneal, limbal, and conjunctival epithelium.

  15. Notch signaling promotes the corneal epithelium wound healing.

    PubMed

    Lu, Huayi; Lu, Qingxian; Zheng, Yajuan; Li, Qiutang

    2012-01-01

    The Notch signaling pathway plays crucial roles in regulation of cell proliferation, differentiation and cell fate decision in multiple tissues and cell types. This study was designed to test the effects of enhanced Notch activity on corneal epithelium homeostasis and wound healing using the transgenic mice that overexpressed an activated Notch1 (NICD) in cornea epithelium. The studies were performed on R26(fN1-ICD) transgenic mice that carry a NICD cDNA (cDNA) whose expression is prevented by a "Lox-STOP-Lox" cassette. When this transgenic mouse is bred to a mouse strain carrying a Cre recombinase expression cassette driven by a tissue-specific keratin 14 (K14) promoter, the floxed "STOP" cassette is excised and NICD is expressed in the cornea epithelium. The expression level of NICD and its downstream target genes, hairy and enhancer of split 1 (Hes1) and hairy/enhancer-of-split related with YRPW motif 1 (Hey1), in the transgenic corneal epithelium was examined by quantitative PCR (qPCR). The phenotypes and morphology of the transgenic corneal epithelium were compared with that of wild type (WT) controls. The proliferation rate of the epithelial cells was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the differentiation statues were examined by K14, tumor protein p63 (p63), K12, and zona occludens 1 (ZO-1) immunoreactivity at either normal developmental condition or after corneal epithelial debridement. The corneal epithelial response to wound healing was studied by fluorescent staining and Richardson's staining macroscopically and by H&E staining at microscope level at 0, 6, 12, 18, and 24 h post injury. Although overexpression of NICD in cornea epithelium led to upregulation of its downstream targets, i.e., Hes1 and Hey1, this did not alter corneal epithelial cell proliferation and differentiation. However, wound healing induced Notch activity and overexpression of NICD promoted corneal epithelial wound healing, which was in agreement with more

  16. Cosmetics Europe multi-laboratory pre-validation of the SkinEthic™ reconstituted human corneal epithelium test method for the prediction of eye irritation.

    PubMed

    Alépée, N; Bessou-Touya, S; Cotovio, J; de Smedt, A; de Wever, B; Faller, C; Jones, P; Le Varlet, B; Marrec-Fairley, M; Pfannenbecker, U; Tailhardat, M; van Goethem, F; McNamee, P

    2013-08-01

    Cosmetics Europe, The Personal Care Association, known as Colipa before 2012, conducted a program of technology transfer and assessment of Within/Between Laboratory (WLV/BLV) reproducibility of the SkinEthic™ Reconstituted Human Corneal Epithelium (HCE) as one of two human reconstructed tissue eye irritation test methods. The SkinEthic™ HCE test method involves two exposure time treatment procedures - one for short time exposure (10 min - SE) and the other for long time exposure (60 min - LE) of tissues to test substance. This paper describes pre-validation studies of the SkinEthic™ HCE test method (SE and LE protocols) as well as the Eye Peptide Reactivity Assay (EPRA). In the SE WLV study, 30 substances were evaluated. A consistent outcome with respect to viability measurement across all runs was observed with all substances showing an SD of less than 18%. In the LE WLV study, 44 out of 45 substances were consistently classified. These data demonstrated a high level of reproducibility within laboratory for both the SE and LE treatment procedures. For the LE BLV, 19 out of 20 substances were consistently classified between the three laboratories, again demonstrating a high level of reproducibility between laboratories. The results for EPRA WLV and BLV studies demonstrated that all substances analysed were categorised similarly and that the method is reproducible. The SkinEthic™ HCE test method entered into the experimental phase of a formal ECVAM validation program in 2010. Copyright © 2013. Published by Elsevier Ltd.

  17. Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision

    NASA Astrophysics Data System (ADS)

    Cursiefen, Claus; Chen, Lu; Saint-Geniez, Magali; Hamrah, Pedram; Jin, Yiping; Rashid, Saadia; Pytowski, Bronislaw; Persaud, Kris; Wu, Yan; Streilein, J. Wayne; Dana, Reza

    2006-07-01

    Transparency of the cornea, the window of the eye, is a prerequisite for vision. Angiogenesis into the normally avascular cornea is incompatible with good vision and, therefore, the cornea is one of the few tissues in the human body where avascularity is actively maintained. Here, we provide evidence for a critical mechanism contributing to corneal avascularity. VEGF receptor 3, normally present on lymphatic and proliferating blood vascular endothelium, is strongly constitutively expressed by corneal epithelium and is mechanistically responsible for suppressing inflammatory corneal angiogenesis. angiogenesis | cornea | lymphatics | inflammation

  18. Estimation of the in vitro eye irritating and inflammatory potential of lipopolysaccharide (LPS) and dust by using reconstituted human corneal epithelium tissue cultures.

    PubMed

    Cao, Yi; Bindslev, Dorthe A; Kjærgaard, Søren K

    2015-01-01

    Eye irritation is a common complaint in indoor environment, but the causes have still not been identified among the multiple exposures in house environments. To identify the potential environmental factors responsible for eye irritation and study the possible mechanisms, an in vitro model for eye irritation is suggested. In this study, reconstituted human corneal epithelium (HCE) tissue cultures were used to study the eye irritating and inflammatory potential of lipopolysaccharide (LPS) and dust. HCE tissue cultures were exposed to a range of concentrations of LPS for 6 h and dust for 24 h, respectively. After exposure, viability and secretion of interleukins (IL) IL-1β, IL-8, and tumor necrosis factor (TNFα) were examined. Histology was used to indicate the morphological changes after dust exposure. Both LPS and dust affected HCE viability. There was an increased level of IL-8 after LPS exposure, while the concentrations of IL-1β and TNFα remained unaffected. Dust exposure resulted in an elevation of both IL-1β and IL-8, but not TNFα. Histology study showed increased vacuolization and reduced thickness after 24 h exposure to 5 mg/mL dust. LPS and dust showed in vitro eye irritating and inflammatory potential, and cytokines/chemokines like IL-1β and IL-8 may be involved in the mechanisms of eye irritation. The HCE tissue culture may be used as an in vitro model to study environmental exposure induced eye irritation and inflammation.

  19. The Effect of Corneal Epithelium on Corneal Curvature in Patients with Keratoconus.

    PubMed

    Akcay, Emine Kalkan; Uysal, Betul Seher; Sarac, Ozge; Ugurlu, Nagehan; Yulek, Fatma; Cagil, Nurullah; Aslan, Nabi

    2015-01-01

    To investigate the effects of corneal epithelium on corneal curvature in patients with keratoconus. This is a prospective, nonrandomized study. Fifty-nine eyes of 47 patients diagnosed as keratoconus and for whom corneal collagen crosslinking (CXL) was recruited in this study. This study is a single-center clinical trial. Pregnancy, lactation, connective tissue disease, corneal thickness below 350 μm, severe dry eyes, or scar of corneal surgery were exclusion criteria. Before and during CXL procedure after removing the corneal epithelium, maximum values of corneal apical curvature, simulated keratometry 1 (Sim-K1), simulated keratometry 2 (Sim-K2), temporal and inferior curvature values, all of which are 1.5 mm from the corneal center, were calculated. These values before and after removal of epithelium were compared statistically. Mean age of patients was 23.30 ± 5.5 (12-38) years. Twenty-eight (59%) were male while 19 (41%) were female. Mean values measured before and after removing the corneal epithelium were: apical curvature; 59.19 ± 7.2 (47.06-82.40) diopter (D) and 61.70 ± 8.8 (49.19-92.66) D (p = 0.001), SimK1; 47.57 ± 4.3 (39.14-64.57) D and 48.23 ± 4.3 (41.89-66.70) D (p = 0.001), SimK2; 52.04 ± 5.3 (43.56-69.34) D and 53.34 ± 5.6 (43.73-70.89) D (p = 0.001), inferior curvature; 53,85 ± 5.2 (43.47-76.56) D and 55.05 ± 5.8 (44.56-81.93) D (p = 0.002), temporal curvature 49.49 ± 5.1 (41.50-71.03) D and 51.53 ± 5.4 (41.58-73.34) D (p = 0.001), respectively. In keratoconus patients during CXL treatment, after removing the corneal epithelium, more steepness is detected in the curvature of the steeper area of the cornea. When evaluating patients with keratoconus, the masking effect of corneal epithelium on values of curvature should be taken into consideration.

  20. Beta adrenoceptors and regenerating corneal epithelium.

    PubMed

    Liu, G S; Trope, G E; Basu, P K

    1990-01-01

    Beta blockers inhibit corneal re-epithelialization. This may be due to beta-2 receptor controlled mechanisms. To investigate this possibility we performed a randomized, double-masked study involving 60 rabbit iatrogenic induced corneal ulcers produced with iodine vapour. Two beta specific drug compounds were tested, namely, betaxolol hydrochloride 0.25% (Alcon) (beta 1) and L132-468 (Sandoz, Basel) 0.25% (beta 2), and phosphate-buffered solution (PBS) as control. There was no statistical difference in the wound healing rates among all groups at 24 hours but there were significant differences at 48 hours (p less than 0.01). At 72 hours, the L132-468 treated groups showed significantly less healing than the betaxolol hydrochloride treated group. The PBS-treated group was healed at this time. By 20th post burning day, SEM revealed that betaxolol hydrochloride treated corneas were completely healed with normal epithelial microvilli. The L132-468 treated corneas were also healed but desquamation and abnormal cells were observed. In conclusion, beta-2 blockers inhibit corneal re-epithelialization more potently than beta-1 blockers.

  1. CON4EI: SkinEthic™ Human Corneal Epithelium Eye Irritation Test (SkinEthic™ HCE EIT) for hazard identification and labelling of eye irritating chemicals.

    PubMed

    Van Rompay, A R; Alépée, N; Nardelli, L; Hollanders, K; Leblanc, V; Drzewiecka, A; Gruszka, K; Guest, R; Kandarova, H; Willoughby, J A; Verstraelen, S; Adriaens, E

    2017-06-15

    Assessment of ocular irritancy is an international regulatory requirement and a necessary step in the safety evaluation of industrial and consumer products. Although a number of in vitro ocular irritation assays exist, none are capable of fully categorizing chemicals as a stand-alone assay. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed with the goal of assessing the reliability of eight in vitro/alternative test methods as well as establishing an optimal tiered-testing strategy. One of the in vitro assays selected was the validated SkinEthic™ Human Corneal Epithelium Eye Irritation Test method (SkinEthic™ HCE EIT). The SkinEthic™ HCE EIT has already demonstrated its capacity to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage (No Category). The goal of this study was to evaluate the performance of the SkinEthic™ HCE EIT test method in terms of the important in vivo drivers of classification. For the performance with respect to the drivers all in vivo Cat 1 and No Cat chemicals were 100% correctly identified. For Cat 2 chemicals the liquids and the solids had a sensitivity of 100% and 85.7%, respectively. For the SkinEthic™ HCE EIT test method, 100% concordance in predictions (No Cat versus No prediction can be made) between the two participating laboratories was obtained. The accuracy of the SkinEthic™ HCE EIT was 97.5% with 100% sensitivity and 96.9% specificity. The SkinEthic™ HCE EIT confirms its excellent results of the validation studies. Copyright © 2017. Published by Elsevier Ltd.

  2. Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells.

    PubMed

    Yin, Ji-Qing; Liu, Wen-Qiang; Liu, Chao; Zhang, Yi-Hua; Hua, Jin-Lian; Liu, Wei-Shuai; Dou, Zhong-Ying; Lei, An-Min

    2013-10-01

    Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.

  3. Deleterious effects of swimming pool chlorine on the corneal epithelium.

    PubMed

    Ishioka, Misaki; Kato, Naoko; Kobayashi, Akira; Dogru, Murat; Tsubota, Kazuo

    2008-01-01

    To study the effect of rinsing with tap and pool water on the ocular surface epithelium. Twenty eyes of 10 volunteers were irrigated in the following order with 250 mL (50 seconds) of physiological salt solution (PSS), distilled water (DW), tap water, or PSS with chlorine (0.5 mg/L). The pH of each fluid was 6.4, 6.8, 6.8, and 6.4, respectively. Vital staining, fluorophotometric assessment, and confocal microscopy were performed before and after irrigation with each fluid. Eyes irrigated with PSS with chlorine showed an increase in fluorescein scores, and eyes washed with both tap water and PSS with chlorine showed an increase in Rose Bengal scores. Corneal fluorescein uptake measured by anterior fluorometer was not altered by eye irrigation with PSS, DW, or tap water. However, PSS with chlorine resulted in a significant increase in corneal fluorescein uptake. Confocal microscopy showed corneal epithelial cell damage in eyes rinsed with PSS with chlorine. Chlorine was determined to be potentially harmful to the corneal epithelial barrier. This study indicated the possibility that swimming without goggles might become a risk factor for corneal epithelial integrity, suggesting encouragement of goggle wear while swimming.

  4. Reconstituted human corneal epithelium: a new alternative to the Draize eye test for the assessment of the eye irritation potential of chemicals and cosmetic products.

    PubMed

    Doucet, O; Lanvin, M; Thillou, C; Linossier, C; Pupat, C; Merlin, B; Zastrow, L

    2006-06-01

    The aim of this study was to evaluate the interest of a new three-dimensional epithelial model cultivated from human corneal cells to replace animal testing in the assessment of eye tolerance. To this end, 65 formulated cosmetic products and 36 chemicals were tested by means of this in vitro model using a simplified toxicokinetic approach. The chemicals were selected from the ECETOC data bank and the EC/HO International validation study list. Very satisfactory results were obtained in terms of concordance with the Draize test data for the formulated cosmetic products. Moreover, the response of the corneal model appeared predictive of human ocular response clinically observed by ophthalmologists. The in vitro scores for the chemicals tested strongly correlated with their respective scores in vivo. For all the compounds tested, the response of the corneal model to irritants was similar regardless of their chemical structure, suggesting a good robustness of the prediction model proposed. We concluded that this new three-dimensional epithelial model, developed from human corneal cells, could be promising for the prediction of eye irritation induced by chemicals and complex formulated products, and that these two types of materials should be tested using a similar protocol. A simple shortening of the exposure period was required for the chemicals assumed to be more aggressively irritant to the epithelial tissues than the cosmetic formulae.

  5. The Effects of Silicone Hydrogel Lens Wear on the Corneal Epithelium and Risk for Microbial Keratitis

    PubMed Central

    Robertson, Danielle M.

    2012-01-01

    Previous studies using animal models and human clinical trials have demonstrated that the use of low oxygen transmissible contact lens materials produce corneal epithelial surface damage resulting in increased Pseudomonas aeruginosa (PA) adhesion and raft-mediated internalization into surface corneal epithelial cells. These findings led to the testable clinical predictions that: (1) microbial keratitis (MK) risk is expected to be greatest during the first 6 months of wear; (2) there is no difference between 6 and 30 night extended wear; and (3) that wear of hyper-oxygen transmissible lenses would reduce the reported incidence of infection. Subsequent epidemiological studies have confirmed the first two predictions; however, increased oxygen transmissibility with silicone hydrogel (SiHy) lens wear has not altered the overall incidence of MK. In this review, more recent clinical and basic studies that investigate epithelial alterations and bacterial adhesion to corneal epithelial cells following wear of SiHy lenses with and without concomitant exposure to chemically preserved multipurpose solutions (MPS) will be examined. The collective results of these studies demonstrate that even in the absence of lens-related hypoxia, MPS induce ocular surface changes during SiHy lens wear which are associated with a pathophysiological increase in PA adherence and internalization in the corneal epithelium, and therefore, predict an increased risk for PA-MK. In addition, new data supporting an interactive role for inflammation in facilitating PA adherence and internalization in the corneal epithelium will also be discussed. PMID:23266590

  6. Cytochrome P450 arachidonic acid metabolism in bovine corneal epithelium

    SciTech Connect

    Masferrer, J.; Schwartzman, M.L.; Abraham, N.G.; Dunn, M.W.; McGiff, J.C.

    1986-03-01

    The presence of the cytochrom P450 system and its involvement in the metabolism of AA was studied in the corneal epithelium. This tissue contains cytochrome P450 as assessed directly by measurement of the carbon monoxide reduced spectrum (specific activity of 161 pmol/10 mg protein) and indirectly by measuring the activity of aryl hydrocarbon hydroxylase (AHH) - a cytochrome P450-dependent enzyme (11-39 pmol 3-OH benzopyrene/mg protein/10 min). When corneal epithelial microsomes were incubated with /sup 14/C-arachidonic acid, 30-50% of the total radioactivity was converted to two peaks, I and II. Further separation using high performance liquid chromatography has shown that each peak contains two metabolites, A,B and C,D. Metabolite formation was dependent on the addition of NADPH (1 mM) and inhibited by carbon monoxide and SKF-525A (100 ..mu..M) suggesting a cytochrome P450-dependent mechanism. Compound C (5-10 ..mu..M) inhibited the activity of corneal epithelial Na-K-ATPase by 30-60%, being 100-fold more potent than ouabain. Compound D (10-100 ng) induced a dose dependent relaxation of the rat caudal artery. Compound D also inhibited corneal Na-K-ATPase activity but less potently than compound C. These compounds may be important to transport processes of ocular epithelia and participate in the control of the ocular circulation and aqueous humor dynamics.

  7. Assessment of size and nucleo-cytoplasmic characteristics of the squamous cells of the corneal epithelium.

    PubMed

    Doughty, Michael J

    2015-05-01

    The aim was to objectively assess size, nucleus and nucleo-cytoplasmic ratio features of squamous cells from the corneal epithelium The corneas of recent post-mortem sheep eyes were either glutaraldehyde-fixed for transmission electron microscopy or impression cytology samples taken, glutaraldehyde-fixed and stained with Giemsa. From the specimens for impression cytology, a representative region was photographed from 12 different samples taken from the central region and 16 different samples taken from mid-peripheral regions of the corneal epithelium. Images were subjected to morphometry after overlays were generated. Electron microscopy revealed a very distinctive stratified corneal epithelium with several superficial layers, confirming the squamous phenotype. Impression cytology from such superficial layers revealed a cell size of 60.1 ± 4.8 μm, nucleus dimension of 12.3 ± 1.5 μm, cell area of 2,419 ± 416 μm(2) and nucleus area of 131 ± 31 μm(2) . A nucleo-cytoplasmic ratio based on nucleus-to-cell length had a mean of 0.207 ± 0.022, while a cytoplasm-to-nucleus length ratio was 3.975 ± 0.474. Estimates of the nucleo-cytoplasmic ratio based on areas had a mean value of 0.059 ± 0.011. Very similar results were found for mid-peripheral corneal epithelium. The results strongly indicate that the squamous phenotype of the superficial corneal epithelial cells is characterised by a large size, large nucleus and low nucleo-cytoplasmic ratio. These morphological characteristics show a notable resemblance to data obtained from impression cytological studies on human conjunctival epithelial cells showing severe squamous metaplasia. © 2015 The Author. Clinical and Experimental Optometry © 2015 Optometry Australia.

  8. [Regeneration of corneal epithelium using keratin modified chitosan membranes].

    PubMed

    Grolik, Maria; Kopeć, Maciej; Szczubiałka, Krzysztof; Wowra, Bogumił; Dobrowolski, Dariusz; Wylegała, Edward; Nowakowska, Maria

    2012-01-01

    The cornea is a transparent front layer of the eye. It functions like a window that controls and focuses the light entering into the eye. The cornea contributes to 65-75% of the eye's total focusing power and it acts as a physical barrier against pathogenic microorganisms, dirt and other noxious physical factors. The corneal tissue is arranged in five basic layers. The outermost layer (epithelium) is made up of highly regenerative cells that allow for quick healing of superficial injuries. Eye infections, diseases, or mechanical injury can harm corneal epithelium and cause blindness. Under certain circumstances, to prevent that, it is recommended to perform complete corneal transplantation. However, due to lack of sufficient number of donors, researchers are searching for alternative solutions.. Regeneration of epidermal tissue can restore and ensure normal functioning of cornea. For that purpose proper grafts are needed. The goal of current research was to develop the material for scaffold preparation providing optimal conditions for the epithelium cornea cell culturing and to determine its chemical, physical, and biological properties. The scaffolds, which could be applied in ophthalmology should fulfill a lot of requirements, among them such as biocompatibility, biodegradability, restorability, non-toxicity. They should also have adequate mechanical strength, flexibility and porosity. The aim of this work was to synthesize and to determine the properties of polymeric material for ophthalmic surgery applications. A hydrogel scaffold in the form of membrane was obtained from chitosan - natural, biocompatible, biologically inert, stable in the natural environmental and antibacterial polysaccharide derived from chitin. Biodegradable chitosan films containing keratin were crosslinked with genipin - a naturally occurring and nontoxic agent. In this study we present physicochemical characterization of the scaffolds. Porosity, contact angle and swelling ratio (at

  9. Role of corneal epithelium in riboflavin/ultraviolet-A mediated corneal cross-linking treatment in rabbit eyes.

    PubMed

    Tao, Xiangchen; Yu, Haiqun; Zhang, Yong; Li, Zhiwei; Jhanji, Vishal; Ni, Shouxiang; Wang, Ya; Mu, Guoying

    2013-01-01

    To evaluate the role of corneal epithelium in riboflavin/ultraviolet-A (UVA) mediated corneal collagen cross-linking treatment. Fifty New Zealand rabbits were divided into 5 groups: UVA treatment with or without corneal epithelium, UVA+riboflavin treatment with or without corneal epithelium, and control without any treatment. All rabbits were sacrificed after irradiation and subsequently 4 mm × 10 mm corneal strips were harvested for biomechanical evaluation. UVA irradiation alone did not enhance the maximal stress and Young's modulus of corneal specimens with (3.15 ± 0.56 mpa, 1.00 ± 0.09 mpa) or without (3.53 ± 0.85 mpa, 0.94 ± 0.21 mpa) the corneal epithelium, compared to specimens in the control group (4.30 ± 0.68 mpa, 1.03 ± 0.24 mpa). However, UVA irradiation combined with riboflavin significantly increased the maximal stress and Young's modulus of corneal specimens with (5.27 ± 1.09 mpa, 1.23 ± 0.23 mpa, P < 0.05) or without (7.16 ± 1.88 mpa, 1.42 ± 0.16 mpa, P < 0.05) corneal epithelium when compared to the control group. The maximal stress and Young's modulus of cornea in UVA+riboflavin and "epithelium-off" group were 35.9% and 15.4% higher compared to the UVA+riboflavin and "epithelium-on" group, respectively (P < 0.05). Our study shows that UVA+riboflavin treatment significantly affects the biomechanical properties of the cornea with and without epithelial removal. However, corneas without epithelium seem to benefit more compared to corneas with the epithelium.

  10. Effects of temperature, amebic strain, and carbohydrates on Acanthamoeba adherence to corneal epithelium in vitro.

    PubMed Central

    Morton, L D; McLaughlin, G L; Whiteley, H E

    1991-01-01

    An in vitro coincubation assay was used to measure adhesion of radiolabeled Acanthamoeba trophozoites to corneal epithelium. Adhesion of amebae to corneal epithelium was higher at 25 degrees C than at 37 or 4 degrees C, did not consistently correlate with the reported pathogenicity of the strain of Acanthamoeba, and was inhibited by mannose and by methyl-alpha-D-mannopyranoside. PMID:1894379

  11. Effects of low-power laser irradiation on the mitosis rate of the corneal epithelium

    NASA Astrophysics Data System (ADS)

    Chen, Varda; Landshman, Nahum; Belkin, Michael

    1995-05-01

    The effect of repeated low power He-Ne laser on rabbit's corneal epithelium was studied after 3 daily sessions. Under certain irradiation parameters, low power He-Ne laser irradiation was found to change the mitotic rate in the basal layer of intact corneal epithelium. Three daily irradiations for 3 or 10 minutes increased the mitotic index while 30 minutes irradiations decreased it.

  12. Electrically assisted delivery of macromolecules into the corneal epithelium.

    PubMed

    Hao, Jinsong; Li, S Kevin; Liu, Chia-Yang; Kao, Winston W Y

    2009-12-01

    Electrically assisted delivery is noninvasive and has been investigated in a number of ocular drug delivery studies. The objectives of this study were to examine the feasibility of electrically assisted delivery of macromolecules such as small interfering RNA (siRNA) into the corneal epithelium, to optimize the iontophoresis and electroporation methods, and to study the mechanisms of corneal iontophoresis for macromolecules. Anodal and cathodal iontophoresis, electroporation and their combinations were the methods examined with mice in vivo. Cyanine 3 (Cy3)-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA and fluorescein isothiocyanate (FITC)-labeled dextran of different molecular weights (4-70 kDa) were the macromolecules studied. Microscopy and histology after cryostat sectioning were used to analyze and compare the delivery of the macromolecules to the cornea. Iontophoresis was effective in delivering siRNA and dextran up to 70 kDa into the cornea. The electroporation method studied was less effective than that of iontophoresis. Although both iontophoresis and electroporation alone can deliver the macromolecules into the cornea, these methods alone were not as effective as the combination of iontophoresis and electroporation (iontophoresis followed by electroporation). The significant enhancement of dextran delivery in anodal iontophoresis suggests that electroosmosis can be a significant flux-enhancing mechanism during corneal iontophoresis. These results illustrate the feasibility of electrically assisted delivery of macromolecules such as siRNA into the cornea.

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

    PubMed Central

    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

  14. Ultrastructural analysis of in vivo expanded corneal epithelium on amniotic membrane.

    PubMed

    Ha, Hyo Shin; Song, Kye Yong; Kim, Jae Chan

    2006-06-01

    The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3+/-1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM.

  15. Ultrastructural Analysis of in vivo Expanded Corneal Epithelium on Amniotic Membrane

    PubMed Central

    Ha, Hyo Shin; Song, Kye Yong

    2006-01-01

    The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3±1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM. PMID:16778403

  16. Wnt/β-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development.

    PubMed

    Zhang, Yujin; Yeh, Lung-Kun; Zhang, Suohui; Call, Mindy; Yuan, Yong; Yasunaga, Mayu; Kao, Winston W-Y; Liu, Chia-Yang

    2015-10-01

    The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear. The Wnt/β-catenin signaling pathway is involved in fundamental aspects of development through the regulation of various growth factors. Here, we show that conditional ablation of either β-catenin (Ctnnb1(cKO)) or co-receptors Lrp5/6 (Lrp5/6(cKO)) in corneal stromal cells results in precocious stratification of the corneal epithelium. By contrast, ectopic expression of a murine Ctnnb1 gain-of-function mutant (Ctnnb1(cGOF)) retards corneal epithelium stratification. We also discovered that Bmp4 is upregulated in the absence of β-catenin in keratocytes, which further triggers ERK1/2 (Mapk3/1) and Smad1/5 phosphorylation and enhances transcription factor p63 (Trp63) expression in mouse corneal basal epithelial cells and in a human corneal epithelial cell line (HTCE). Interestingly, mouse neonates given a subconjunctival BMP4 injection displayed a phenotype resembling that of Ctnnb1(cKO). Conditional ablation of Bmp4 eradicates the phenotype produced in Ctnnb1(cKO) mice. Furthermore, ChIP and promoter-luciferase assays show that β-catenin binds to and suppresses Bmp4 promoter activity. These data support the concept that cross-talk between the Wnt/β-catenin/Bmp4 axis (in the stromal mesenchyme) and Bmp4/p63 signaling (in the epithelium) plays a pivotal role in epithelial stratification during corneal morphogenesis.

  17. Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium.

    PubMed

    Baiocchi, Stefano; Mazzotta, Cosimo; Cerretani, Daniela; Caporossi, Tomaso; Caporossi, Aldo

    2009-05-01

    To evaluate intrastromal concentrations of riboflavin with and without epithelium to ensure the efficacy and safety of corneal crosslinking (CXL) by the standard and transepithelial procedures. Department of Ophthalmology and Department of Pharmacology G. Segre, Siena University, Siena, Italy. This study comprised keratoconic patients enrolled for penetrating keratoplasty (PKP) and warm-stored sclerocorneal rings unsuitable for transplantation. Half the PKP specimens were debrided, and half were left with the epithelium in situ. One of the latter and 1 debrided sample were not exposed to riboflavin (controls). Samples in both groups were soaked with 0.1% riboflavin-dextran 20% solution instilled every 2 minutes for 5, 15, and 30 minutes. Riboflavin concentrations were determined by high-performance liquid chromatography (HPLC). The study evaluated 14 PKP specimens and 16 sclerocorneal rings. Control samples did not show a riboflavin emission peak. In exposed samples with epithelium, the mean riboflavin concentration was 91.88 ng/g after 5 minutes of exposure, 95.60 ng/g after 15 minutes, and 94.92 ng/g after 30 minutes. In the debrided samples, the mean riboflavin concentration was 14.42 microg/g, 20.92 microg/g, and 24.06 microg/g, respectively. No differences were seen between the in vivo samples and the ex vivo samples. The HPLC quantitative study showed that stromal concentrations of riboflavin increased with exposure time only if the epithelium was removed. A theoretically safe and effective riboflavin concentration of 15 microg/g was obtained for ultraviolet A-induced CXL only after the epithelium was removed and after at least 10 minutes of riboflavin application every 2 minutes.

  18. NC-1059: a channel-forming peptide that modulates drug delivery across in vitro corneal epithelium.

    PubMed

    Martin, Jesica; Malreddy, Pradeep; Iwamoto, Takeo; Freeman, Lisa C; Davidson, Harriet J; Tomich, John M; Schultz, Bruce D

    2009-07-01

    The goal of this study was to determine whether a synthetic peptide, NC-1059, can modulate the corneal epithelium to increase the permeation of therapeutic agents across this barrier. An in vitro system employing transformed human corneal epithelial (THCE) cells was optimized for this study. Culture conditions were identified to promote formation of a confluent monolayer that rapidly develops a substantial transepithelial electrical resistance. Electrical parameters were measured with a modified Ussing flux chamber, and solute flux was quantified with fluorescently labeled compounds. NC-1059 causes a concentration-dependent increase in short-circuit current and an increase in transepithelial electrical conductance when assessed in modified Ussing chambers. The effect of NC-1059 on transepithelial electrical resistance was reversible. To test for paracellular permeability and size exclusion, FITC-labeled dextran ranging in size from 10 to 70 kDa was used. Dextran permeated the corneal cell monolayer in the presence, but not the absence, of NC-1059. Fluorescein sodium and carboxyfluorescein were then used as low molecular weight markers with similar NC-1059-modulated kinetics being observed. Maximum permeation for the fluorescein derivatives occurred 30 to 90 minutes after a 5-minute NC-1059 exposure. A prototypical drug, methotrexate, also exhibited increased permeation in the presence of NC-1059. NC-1059 enhances drug permeation across cultured corneal epithelial cell monolayers by transiently affecting the paracellular pathway. Thus, NC-1059 is a lead compound for development of cotherapeutic agents to enhance access and effectiveness of ophthalmic compounds.

  19. Signature microRNAs in human cornea limbal epithelium.

    PubMed

    Teng, Yufei; Wong, Hoi Kin; Jhanji, Vishal; Chen, Jian Huan; Young, Alvin Lerrmann; Zhang, Mingzhi; Choy, Kwong Wai; Mehta, Jodhbir Singh; Pang, Chi Pui; Yam, Gary Hin-Fai

    2015-05-01

    This study was aimed to identify the signature microRNAs, which regulate the biological processes of corneal epithelial progenitor cell (CEPC) homeostasis and regulation through characterizing the differential expression profile of microRNAs in human limbal epithelium containing adult CEPC versus central corneal epithelium without CEPC. MicroRNA microarray had identified 37 microRNAs enriched in human corneal epithelium. Among them, nine were significantly upregulated in limbal epithelium and one in central corneal epithelium after validation by TaqMan® real-time polymerase chain reaction. In addition to our previous finding of miR-143 and 145, the expression of miR-10b, 126, and 155 was localized in limbal epithelium (LE) (predominantly basal layers) by using locked nucleic acid-based in situ hybridization. Potential target genes were predicted by TargetScan Human v6.0 and compared to the reported human cornea epithelial gene profile GSE5543. Analyzed by web-based Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and DAVID Functional Annotation Bioinformatics Resources v6.7, the downregulated genes were involved in pathways of immune response and cellular protection, apoptosis, and cell movement whereas upregulated genes with cell survival, cell-matrix interaction, and cell-cell adhesion. We found a constant occurrence of miR-143, 145, and 155 in all KEGG pathways regulating limbal epithelial events. By Ingenuity Systems (IPA®) analysis, these microRNAs could cooperatively regulate cell growth and apoptosis via tumor necrosis factor activation and MYC repression. Our findings thus suggest a unique microRNA signature existing in human limbal epithelium and participating in CEPC homeostasis.

  20. Thermosensitive chitosan-based hydrogels releasing stromal cell derived factor-1 alpha recruit MSC for corneal epithelium regeneration.

    PubMed

    Tang, Qiaomei; Luo, Chenqi; Lu, Bing; Fu, Qiuli; Yin, Houfa; Qin, Zhenwei; Lyu, Danni; Zhang, Lifang; Fang, Zhi; Zhu, Yanan; Yao, Ke

    2017-10-01

    Corneal epithelium integrity depends on continuous self-renewing of epithelium and connections between adjacent cells or between the cells and the basement membrane. Self-renewing epithelium cells mainly arise from the continuous proliferation and differentiation of the basal layer and limbal stem cells. The aim of the present study was to generate a bioactive, thermosensitive chitosan-gelatin hydrogel (CHI hydrogel) by incorporating exogenous recombinant human stromal cell-derived factor-1 alpha (SDF-1 alpha) for corneal epithelium regeneration. The exogenous SDF-1 alpha could enhance the stem cells proliferation, chemotaxis and migration, and the expression levels of related genes were significantly elevated in LESCs and mesenchymal stem cells (MSCs) in vitro. Moreover, the MSCs promoted the proliferation and maintained the corneal fate of the LESCs. The rat alkali injury model was used for in vivo study. The injured eyes were covered with CHI hydrogel alone or rhSDF-1 alpha-loaded CHI hydrogel. All rats were followed for 13days. Histological examination showed that the SDF-1 alpha/CHI hydrogel complex group had a nearly normal thickness; moreover, it was also found that this group could upregulate the expression of some genes and had more ΔNp63-positive cells. The SDF-1 alpha/CHI hydrogel complex group had a more tightly arranged epithelium compared with the control group using transmission electron microscopy (TEM). The mechanism for this may have involved the activation of stem cell homing and the secretion of growth factors via the SDF-1/CXCR4 chemokine axis. Therefore, SDF-1 alpha/CHI hydrogel complexes could provide a new idea for the clinical application. The clarity of cornea is important for normal vision. The loss or dysfunction of LESCs leads to the impairment of corneal epithelium. The complete regeneration of corneal epithelium has not been achieved. Our study demonstrated that the incorporation of rhSDF-1 alpha with CHI hydrogel accelerated corneal

  1. In vitro 3D corneal tissue model with epithelium, stroma, and innervation.

    PubMed

    Wang, Siran; Ghezzi, Chiara E; Gomes, Rachel; Pollard, Rachel E; Funderburgh, James L; Kaplan, David L

    2017-01-01

    The interactions between corneal nerve, epithelium, and stroma are essential for maintaining a healthy cornea. Thus, corneal tissue models that more fully mimic the anatomy, mechanical properties and cellular components of corneal tissue would provide useful systems to study cellular interactions, corneal diseases and provide options for improved drug screening. Here a corneal tissue model was constructed to include the stroma, epithelium, and innervation. Thin silk protein film stacks served as the scaffolding to support the corneal epithelial and stromal layers, while a surrounding silk porous sponge supported neuronal growth. The neurons innervated the stromal and epithelial layers and improved function and viability of the tissues. An air-liquid interface environment of the corneal tissue was also mimicked in vitro, resulting in a positive impact on epithelial maturity. The inclusion of three cell types in co-culture at an air-liquid interface provides an important advance for the field of in vitro corneal tissue engineering, to permit improvements in the study of innervation and corneal tissue development, corneal disease, and tissue responses to environmental factors.

  2. Galectin-3 enhances extracellular matrix associations and wound healing in monkey corneal epithelium.

    PubMed

    Fujii, Atsuko; Shearer, Thomas R; Azuma, Mitsuyoshi

    2015-08-01

    Poor healing of epithelial wounds in cornea is a major clinical problem, leading to persistent epithelial defects and ulceration. The primary cause is poor cell migration over the wound. Carbohydrate-binding protein galectin-3 binds to extracellular matrixes (ECMs) and promotes lamellipodia formation by cross-linking to α3 integrin. Recombinant galectin-3 also facilitates wound healing in the rodent cornea. The purposes of the present experiments were to: (1) establish epithelial wound healing models in monkey corneal explant culture, the models more relevant to human, (2) evaluate the healing effect of galectin-3 in our models, and (3) determine if galectin-3 enhances cell adhesion by interacting with ECMs on corneal surface and their ligand integrins. Monkey corneas with central wounds produced by sodium hydroxide (NaOH) or n-heptanol were incubated with or without recombinant galectin-3. The defected area was stained with sodium fluorescein. Primary isolated corneal epithelial cells from monkey were cultured with or without galectin-3 on plates coated with ECMs or integrins, and the number of adhering cells was counted. Galectin-3 expression in various eye tissues was visualized by immunoblotting. NaOH caused loss of epithelial cells and basement membrane. n-Heptanol removed epithelial cells, but the basement membrane was retained. These corneal defects spontaneously became smaller in a time-dependent manner. Exogenous galectin-3 enhanced wound healing in both NaOH and n-heptanol models. Galectin-3 also enhanced cell adhesion onto the major ECMs found in the basement and Bowman's membranes and onto integrins. Relatively high levels of galectin-3 were detected in corneal and conjunctival epithelium, but tear fluid contained negligible galactin-3. These results suggested that the enhanced binding of epithelial cells to ECMs and integrins caused by galectin-3 might promote cell migration over wounded corneal surfaces. Since tear fluid contained relatively low

  3. Location of penetration and metabolic barriers to levobunolol in the corneal epithelium of the pigmented rabbit.

    PubMed

    Ashton, P; Wang, W; Lee, V H

    1991-11-01

    The objective of this study was to determine which of the five or six corneal epithelial layers was rate-limiting in the corneal penetration and metabolism of levobunolol in the pigmented rabbit. Corneal penetration and metabolism were evaluated using the isolated cornea in the modified Ussing chamber. Levobunolol and its metabolite, dihydrolevobunolol, were assayed by reversed phase high-performance liquid chromatography using spectrophotometric detection. EDTA (0.1 and 0.5%) and benzalkonium chloride (0.005-0.05%) were used to disrupt the integrity of the corneal epithelial layers. EDTA, which loosened the tight junctions between the superficial corneal epithelial cells, reduced both the transcorneal flux and metabolism of levobunolol. In contrast, benzalkonium chloride, which disrupted the integrity of the outermost corneal epithelial layers, enhanced the transcorneal levobunolol flux while reducing its extent of metabolism. The extent of enhancement in transcorneal flux afforded by 0.025% benzalkonium chloride was comparable to that seen in the deepithelized cornea. Within 5 min of contact by the corneal epithelium with this preservative, the ratio of dihydrolevobunolol concentration on the endothelial to the epithelial side was reduced by two-thirds. Although direct confirmation is required, the above findings are consistent with the hypothesis that the rate-limiting layer to corneal penetration of levobunolol resides in the outermost two to three layers of the corneal epithelium, whereas the metabolic barrier resides in deeper lying regions.

  4. Deletion of JAM-A causes morphological defects in the corneal epithelium.

    PubMed

    Kang, Liang I; Wang, Yan; Suckow, Arthur T; Czymmek, Kirk J; Cooke, Vesselina G; Naik, Ulhas P; Duncan, Melinda K

    2007-01-01

    Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.

  5. Culture of the primary corneal epithelium as a potential component of test batteries for eye irritancy testing.

    PubMed

    Sladowski, D; Liberek, I; Lipski, K; Ozga, T; Olkowska-Truchanowicz, J; Szaflik, J

    2005-10-01

    One of the main goals for toxicologists working on the development of in vitro tests is to replace the animal-based eye irritation test. Inflammation is one of the mechanisms which have not been covered sufficiently by the existing in vitro ocular irritancy test systems. As there are major species differences between the human and rabbit eye inflammation mechanisms, the most relevant test system is the human eye itself. The current study focused on an evaluation of the practical availability of human corneal epithelial cells for routine eye irritancy testing. Human corneal epithelium cell cultures were used to assess the effects of lipopolysaccharide on IL-1 beta release. The findings indicated that cytokine release can be augmented by the presence of the complement system, which is normally found in tears. However, the corneal cells were found to be highly resistant to the complement system, which can be attributed to the very high expression of CD59, a powerful complement regulatory protein found in the corneal epithelium. It is estimated that discarded corneas from tissue banks could provide enough material for routine testing by this method.

  6. Multipurpose Care Solution–Induced Corneal Surface Disruption and Pseudomonas aeruginosa Internalization in the Rabbit Corneal Epithelium

    PubMed Central

    Posch, Leila C.; Zhu, Meifang; Robertson, Danielle M.

    2014-01-01

    Purpose. To evaluate the effects of a chemically preserved multipurpose contact lens care solution (MPS) on the corneal epithelial surface and Pseudomonas aeruginosa (PA) internalization in the rabbit corneal epithelium. Methods. Rabbits were fit in one eye with a silicone hydrogel lens (balafilcon A) soaked overnight in a borate-buffered MPS (BioTrue). The contralateral eye was fit with a lens removed directly from the blister pack containing borate-buffered saline (control). Lenses were worn for 2 hours. Upon lens removal, corneas were challenged ex vivo with invasive PA strain 6487 and assessed for PA internalization. Ultrastructural changes were assessed using scanning electron (SEM) and transmission electron microscopy (TEM). Results. Scanning electron microscopy showed frank loss of surface epithelium in MPS-exposed eyes, while control eyes exhibited occasional loss of surface membranes but retention of intact junctional borders. Transmission electron microscopy data supported and extended SEM findings, demonstrating the presence of epithelial edema in MPS-treated eyes. There was a 12-fold increase in PA uptake into the corneal epithelium following wear of the MPS-treated lens compared to control (P = 0.008). Conclusions. These data demonstrate that corneal exposure to MPS during lens wear damages the surface epithelium and are consistent with our previous clinical data showing an increase in bacterial binding to exfoliated epithelial cells following MPS use with resultant increased risk for lens-mediated infection. These findings also demonstrate that the PA invasion assay may provide a highly sensitive quantitative metric for assessing the physiological impact of lens-solution biocompatibility on the corneal epithelium. PMID:24876286

  7. Effect of the synthetic NC-1059 peptide on diffusion of riboflavin across an intact corneal epithelium.

    PubMed

    Zhang, Yuntao; Sukthankar, Pinakin; Tomich, John M; Conrad, Gary W

    2012-05-04

    To investigate the effect of the peptide NC-1059 on riboflavin (RF) diffusion across an intact corneal epithelium into the stroma. NC-1059 peptide was synthesized by solid-phase synthesis with 9-fluorenylmethoxycarbonyl chemistry, characterized by reversed-phase HPLC, and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. The diffusion of RF across embryonic day 18 chick corneal epithelium ex vivo was monitored using confocal microscopy. The depth distributions of RF in the corneal stroma were calculated using a group of linear equations based on the relationship between RF fluorescence intensity and concentration. Data presented in this study demonstrate that the NC-1059 peptide can transiently open the intact epithelial barrier to allow the permeation of RF into the stroma. The effect of NC-1059 peptide on RF diffusion across the corneal epithelium was concentration and time dependent. The amount of RF reaching a 50-μm depth of chick corneal stoma increased dramatically after exposure to NC-1059 for 10 minutes, reaching a plateau by 30 minutes. The concentrations of RF in the presence of NC-1059 at corneal stromal depths of 50, 100, and 150 μm were significantly higher than in the absence of the peptide, and almost as high as in corneas in which the epithelium first had been physically removed. In addition, a cell viability assay indicated that the NC-1059 peptide did not kill corneal epithelial cells. NC-1059 peptide significantly enhances the diffusion of RF across intact corneal epithelium into the stroma.

  8. Hypoxia, overnight wear, and tear stagnation effects on the corneal epithelium: data and proposed model.

    PubMed

    Lin, Meng C; Polse, Kenneth A

    2007-11-01

    : To explore the possible mechanisms that may lead to overnight contact lens-associated corneal morbidity by examining data from several corneal epithelial permeability experiments obtained under different environmental and lens fitting paradigms. : Epithelial permeability was assessed by using fluorometry to determine the fluorescein penetration rate from the tear film into the corneal stroma. Changes in this rate provide an index of the corneal epithelial status; increased permeability leads to decreased barrier function. : Hypoxia and tear stagnation during overnight lens wear play a significant role in altering the corneal epithelial barrier function (P<0.05). However, eliminating lens-induced hypoxia alone does not ameliorate changes in epithelial status (P<0.05). : Based on data from these experiments, it is suggested that hypoxia and tear stagnation should be eliminated to minimize alteration of the corneal epithelium associated with overnight contact lens wear.

  9. Regeneration of the corneal epithelium after debridement of its central region: an autoradiographic study on rabbits.

    PubMed

    Barbosa, Flávia Leão; Góes, Rejane Maira; de Faria-E-Sousa, Sidney Júlio; Haddad, Antonio

    2009-08-01

    To investigate the proliferative behavior of the corneal and limbal epithelia after debridement on the central region of the rabbit cornea. After scraping a circular epithelial area, 5 mm in diameter, in the center of the cornea, ([3]) H-thymidine ( ([3]) H-TdR) was injected intravitreally, and the rabbits killed from 1 to 49 days afterward. The cornea, together with the adjacent conjunctiva, was processed for autoradiography. The regenerating epithelium at the center of the cornea exhibited high frequencies of labeled nuclei when compared to controls. The mitotic indexes for the limbus were comparable in experimental and control eyes. The unique basal stratum of the limbal epithelium exhibited quick proliferation and vertical migration in all eyes. Cells that remained labeled for four weeks or more were observed throughout the corneal epithelium, including its basal stratum, and this did not depend on epithelial damage. Corneal epithelium wounds are healed by sliding and proliferation of cells surrounding the epithelial gap without any evidence for the participation of the limbal epithelium. Daughter cells labeled with ([3]) H-TdR were visualized in all layers of the corneal epithelium up to 7 weeks after the DNA precursor injection. However, at this long interval, the only labeled cells in the limbus were in the suprabasal layers.

  10. THE GOLGI APPARATUS IN CHICK CORNEAL EPITHELIUM: CHANGES IN INTRACELLULAR POSITION DURING DEVELOPMENT

    PubMed Central

    Trelstad, Robert L.

    1970-01-01

    The intracellular position of the Golgi apparatuses in the basal cell layer of the corneal epithelium in embryonic and hatched chicks has been studied in the light microscope by impregnating the Golgi apparatus with silver. During two distinct periods in development the Golgi apparatuses in the basal cells shift from an apical to basal position. Each of these periods correlates in time with the appearance of an acellular collagenous matrix beneath the epithelium. Examination of the basal epithelial cells in the electron microscope confirms the intracellular shifts in position of the Golgi apparatus. The results suggest that the Golgi apparatus shifts to the basal cell pole of the corneal epithelium in order to excrete connective tissue materials into the developing corneal stroma. PMID:4195852

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

    SciTech Connect

    Morishige, Naoyuki; Ko, Ji-Ae; Morita, Yukiko; Nishida, Teruo

    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

  12. Hypoxia-induced downregulation of ΔNp63α in the corneal epithelium

    PubMed Central

    Robertson, Danielle M.; Zhu, Meifang; Wu, Yu-Chieh; Cavanagh, H. Dwight

    2012-01-01

    Purpose To establish a relationship between hypoxic stress and the expression of ΔNp63α in an established rabbit contact lens model and in cultured corneal epithelial cells. Methods New Zealand White rabbits were fit in one eye with either a non-oxygen transmissible or hyper oxygen permeable rigid contact lens for 24 hours of wear; the contralateral eye was used as a control. All rabbits underwent a bilateral nictitating membranectomy to facilitate lens retention. ΔNp63α expression was analyzed by immunofluorescence and western blot. Telomerase-immortalized human corneal epithelial cells (hTCEpi) were grown in serum-free media and treated with the hypoxia mimetic cobalt chloride to simulate hypoxia for 6 (short term) or 24 (prolonged) hours. Transcriptional activity and protein levels were assessed using luciferase reporter assays, RT-PCR, and western blot. Cell viability was assessed by live/dead assay. Results Compared to the non-lens wearing eye, 24 hours of non-oxygen transmissible lens wear in vivo decreased ΔNp63α protein levels in both the limbal and central corneal epithelium; this decrease was not found in the hyper oxygen transmissible lens group. In hTCEpi cells in vitro, hypoxia increased the activity of the ΔN promoter, but reduced levels of ΔNp63α mRNA after 24 hours of prolonged culture. Similarly, ΔNp63α expression levels were unaffected from short term exposure, but decreased after 24 hours. Live/dead assay confirmed the presence of viable cells following CoCl2 treatment at 6 and 24 hour time points. Cells treated for 24 hours were viable but were smaller and rounded with signs of membrane blebbing, consistent with early stages of apoptosis. Conclusions Hypoxic stress induced by either prolonged wear of a non-oxygen transmissible lens in vivo or hypoxic-mimic conditions by cobalt chloride in vitro down regulates ΔNp63α in the corneal epithelium. The loss of ΔNp63α in response to hypoxic stress may contribute to the disruption of

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

    PubMed

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

    2006-06-01

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

  14. Evaluation of the cytotoxic effects of ophthalmic solutions containing benzalkonium chloride on corneal epithelium using an organotypic 3-D model

    PubMed Central

    Khoh-Reiter, Su; Jessen, Bart A

    2009-01-01

    Background Benzalkonium chloride (BAC) is a common preservative used in ophthalmic solutions. The aim of this study was to compare the cytotoxic effects of BAC-containing ophthalmic solutions with a BAC-free ophthalmic solution using an organotypic 3-dimensional (3-D) corneal epithelial model and to determine the effects of latanoprost ophthalmic solution and its BAC-containing vehicle on corneal thickness in a monkey model. Methods The cytotoxicity of commercially available BAC-containing ophthalmic formulations of latanoprost (0.02% BAC) and olopatadine (0.01% BAC) was compared to that of BAC-free travoprost and saline in a corneal organotypic 3-D model using incubation times of 10 and 25 minutes. To compare the extent of differentiation of 3-D corneal cultures to monolayer transformed human corneal epithelial (HCE-T) cell cultures, expression levels (mRNA and protein) of the corneal markers epidermal growth factor receptor, transglutaminase 1 and involucrin were quantified. Finally, latanoprost ophthalmic solution or its vehicle was administered at suprapharmacologic doses (two 30 μL drops twice daily in 1 eye for 1 year) in monkey eyes, and corneal pachymetry was performed at baseline and at weeks 4, 13, 26 and 52. Results In the 3-D corneal epithelial culture assays, there were no significant differences in cytotoxicity between the BAC-containing latanoprost and olopatadine ophthalmic solutions and BAC-free travoprost ophthalmic solution at either the 10- or 25-minute time points. The 3-D cultures expressed higher levels of corneal epithelial markers than the HCE-T monolayers, indicating a greater degree of differentiation. There were no significant differences between the corneal thickness of monkey eyes treated with latanoprost ophthalmic solution or its vehicle (both containing 0.02% BAC) and untreated eyes. Conclusion The lack of cytotoxicity demonstrated in 3-D corneal cultures and in monkey studies suggests that the levels of BAC contained in ophthalmic

  15. Identification of striatin, a desmosomal protein, in the canine corneal epithelium.

    PubMed

    Stern, Joshua A; Lahmers, Sunshine; Meurs, Kathryn M

    2015-10-01

    Striatin is a scaffolding protein expressed in brain and cardiac tissues. In the heart, striatin has been localized to the region of the cardiac desmosome. A causal mutation within the gene encoding for this scaffolding protein has been described as the etiology for arrhythmogenic right ventricular cardiomyopathy, a disease of the cardiac desmosome, in a canine model. Hemidesmosomes are cell adhesion complexes located within the cornea where they anchor the corneal epithelium to the stroma at the basement membrane and participate in cell-signaling processes. Traditional cell adhesion desmosomes are also known to link the corneal epithelial cells together. We hypothesized that striatin may be found in the cornea localized to regions of either hemidesmosomes and/or desmosomes. Immunohistochemical evaluation was performed to evaluate for striatin labeling in normal canine cornea. Striatin was localized to the cytoplasmic region of corneal epithelial cells. The role of striatin in corneal disease warrants investigation.

  16. Sensory and sympathetic innervation of the mouse and guinea pig corneal epithelium.

    PubMed

    Ivanusic, Jason J; Wood, Rhiannon J; Brock, James A

    2013-03-01

    This study used immunohistochemistry, retrograde tracing, and high-resolution confocal microscopy to explore the structure and neurochemistry of nerve terminals in the corneal epithelium of mice and guinea pigs. In both species, sub-basal nerves formed a plexus in the basal epithelium. Some axons had bulbar endings within the basal epithelium, but most projected perpendicularly from sub-basal nerves to within a few micrometers of the epithelial surface. Three morphologies for these nerve terminals were identified. Simple terminals did not branch after leaving the sub-basal nerves and ended with a single, bulbar swelling. Ramifying terminals branched in the squamous cell layer, forming horizontal fibers that ran parallel to the surface and terminated with single bulbar swellings. Complex terminals branched as they approached the epithelial surface, forming a cluster of highly branched fibers with multiple bulbar endings. Calcitonin gene-related peptide immunolabeled (peptidergic) axons ended mostly in simple terminals, whereas transient receptor potential cation channel subfamily M member 8 immunolabeled (cold receptor) axons ended almost exclusively in complex terminals. Retrograde labeling identified discrete subpopulations of corneal afferent neurons in the trigeminal ganglion. Tyrosine hydroxylase-immunolabeled (sympathetic) nerve terminals originating from the superior cervical ganglion occurred throughout the corneal epithelium of mice, but only in the basal epithelium of guinea pigs. These findings demonstrate that nerve terminals in the corneal epithelium of mice and guinea pigs can be distinguished on the basis of their morphology and neurochemistry, and suggest that nerve terminals with different sensory modalities can be defined on the basis of their morphology.

  17. 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

    PubMed Central

    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

  18. Localization and Expression of Zonula Occludins-1 in the Rabbit Corneal Epithelium following Exposure to Benzalkonium Chloride

    PubMed Central

    Zhang, Zhenhao; Chen, Lelei; Xie, Hui; Dong, Nuo; Chen, Yongxiong; Liu, Zuguo

    2012-01-01

    Preservatives are a major component of the ophthalmic preparations in multi-dose bottles. The purpose of this study was to investigate the acute effect of benzalkonium chloride (BAC), a common preservative used in ophthalmic preparations, on the localization and expression of zonula occludens (ZO)-1 in the rabbit corneal epithelium in vivo. BAC at 0.005%, 0.01%, or 0.02% was topically applied to one eye each of albino rabbits at 5 min intervals for a total of 3 times. The contralateral untreated eyes served as controls. The following clinical indications were evaluated: Schirmer test, tear break-up time (BUT), fluorescein and rose Bengal staining. The structure of central cornea was examined by in vivo confocal microscopy, and the corneal barrier function was evaluated by measurement of corneal transepithelial electrical resistance and permeability to carboxy fluorescein. Whole mount corneas were analyzed by using fluorescence confocal microscopy for the presence of ZO-1, 2, occludin, claudin-1, Ki67 and cell apoptosis in the epithelium. The expression of ZO-1 in the corneal epithelium was also examined by western blot and reverse transcription-polymerase chain reaction analyses. Exposure to BAC resulted in higher rose Bengal staining scores while no significant changes in BUT, Schirmer and corneal florescein scores. It also induced corneal epithelial cell damage, dispersion of ZO-1 and ZO-2 from their normal locus at the superficial layer and disruption of epithelial barrier function. However, the amounts of ZO-1 mRNA and protein in the corneal epithelium were not affected by BAC treatment. Exposure to BAC can quickly impair the corneal epithelium without tear deficiency. BAC disrupts the tight junctions of corneal epithelium between superficial cells in the rabbit corneal epithelium in vivo. PMID:22815857

  19. Localization and expression of zonula occludins-1 in the rabbit corneal epithelium following exposure to benzalkonium chloride.

    PubMed

    Chen, Wensheng; Hu, Jiaoyue; Zhang, Zhenhao; Chen, Lelei; Xie, Hui; Dong, Nuo; Chen, Yongxiong; Liu, Zuguo

    2012-01-01

    Preservatives are a major component of the ophthalmic preparations in multi-dose bottles. The purpose of this study was to investigate the acute effect of benzalkonium chloride (BAC), a common preservative used in ophthalmic preparations, on the localization and expression of zonula occludens (ZO)-1 in the rabbit corneal epithelium in vivo. BAC at 0.005%, 0.01%, or 0.02% was topically applied to one eye each of albino rabbits at 5 min intervals for a total of 3 times. The contralateral untreated eyes served as controls. The following clinical indications were evaluated: Schirmer test, tear break-up time (BUT), fluorescein and rose Bengal staining. The structure of central cornea was examined by in vivo confocal microscopy, and the corneal barrier function was evaluated by measurement of corneal transepithelial electrical resistance and permeability to carboxy fluorescein. Whole mount corneas were analyzed by using fluorescence confocal microscopy for the presence of ZO-1, 2, occludin, claudin-1, Ki67 and cell apoptosis in the epithelium. The expression of ZO-1 in the corneal epithelium was also examined by western blot and reverse transcription-polymerase chain reaction analyses. Exposure to BAC resulted in higher rose Bengal staining scores while no significant changes in BUT, Schirmer and corneal florescein scores. It also induced corneal epithelial cell damage, dispersion of ZO-1 and ZO-2 from their normal locus at the superficial layer and disruption of epithelial barrier function. However, the amounts of ZO-1 mRNA and protein in the corneal epithelium were not affected by BAC treatment. Exposure to BAC can quickly impair the corneal epithelium without tear deficiency. BAC disrupts the tight junctions of corneal epithelium between superficial cells in the rabbit corneal epithelium in vivo.

  20. Differentiation of embryonic stem cells into corneal epithelium.

    PubMed

    Wang, Zhichong; Ge, Jian; Huang, Bing; Gao, Qianying; Liu, Bingqian; Wang, Linghua; Yu, Ling; Fan, Zhigang; Lu, Xiaoming; Liu, Jingbo

    2005-10-01

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

  1. Invisible Shield: Review of the Corneal Epithelium as a Barrier to UV Radiation, Pathogens, and Other Environmental Stimuli.

    PubMed

    Bashir, Hasan; Seykora, John T; Lee, Vivian

    2017-01-01

    The ocular surface is comprised of the cornea and conjunctiva, which are structures that not only protect the eye but also enable vision. The corneal epithelium is the most superficial layer of the cornea, and therefore first line of defense against external assaults. Damage to this highly specialized structure could lead to vision loss, making it an important structure to investigate and understand. Here, we conducted a search of the current literature on the mechanisms the corneal epithelium has adapted against three frequent insults: UV-radiation, pathogens, and environmental assaults. This review systematically examines the corneal epithelium's response to each assault in order to maintain its role as an invisible shield. The goal of this review is to provide insight into some of the critical functions the corneal epithelium performs that may be valuable to current regenerative studies.

  2. [The artificial epithelium in chronic corneal diseases and to avoid emergency keratoplasty (author's transl)].

    PubMed

    Turss, R; Retzlaff, K; Hebrock, R

    1979-02-01

    Report on the treatment of 41 patients in the last 10 years. In chronic corneal diseases epikeratoprosthesis is possible when every other therapy failed. With growing experience functional results are better and complications seldom. Since several years we use glued-on contact lenses in acute ulcers too in order to avoid emergency keratoplasty. When suitable donor material is missing or if plastic surgery of the eye lids is necessary the artificial epithelium prevents ulcer perforation as a mechanical collagenase inhibitor. The anterior chamber can be reinstalled in perforated ulcers by sealing with cyanoacrylate glue and covering with artificial epithelium. A corticosteroid therapy of the iritis becomes possible to avoid the frequent complication of anterior synechia in later keratoplasty. By reducing the steroid dosis vascularisation of the ulcer is reached and a corneal grafting can be evaded sometimes if the prognosis of keratoplasty is poor or the central cornea is clear such as in ulcers near the limbus.

  3. An Ultra-thin Amniotic Membrane as Carrier in Corneal Epithelium Tissue-Engineering.

    PubMed

    Zhang, Liying; Zou, Dulei; Li, Sanming; Wang, Junqi; Qu, Yangluowa; Ou, Shangkun; Jia, Changkai; Li, Juan; He, Hui; Liu, Tingting; Yang, Jie; Chen, Yongxiong; Liu, Zuguo; Li, Wei

    2016-02-15

    Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 μm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.

  4. The Acetylcholine Signaling Network of Corneal Epithelium and Its Role in Regulation of Random and Directional Migration of Corneal Epithelial Cells

    PubMed Central

    Chernyavsky, Alex I.; Galitovskiy, Valentin; Shchepotin, Igor B.; Jester, James V.; Grando, Sergei A.

    2014-01-01

    Purpose. Because cholinergic drugs are used in ophthalmology and cholinergic stimulation has been shown to facilitate epithelialization of mucocutaneous wounds, we performed a systematic analysis of components of the cholinergic network of human and murine corneal epithelial cells (CECs) and determined the role of autocrine and paracrine acetylcholine (ACh) in regulation of CEC motility. Methods. We investigated the expression of ACh receptors at the mRNA and protein levels in human immortalized CECs, localization of cholinergic molecules in normal and wounded murine cornea, and the effects of cholinergic drugs on CEC directional and random migration in vitro, intercellular adhesion, and expression of integrin αV and E-cadherin. Results. We demonstrated that corneal epithelium expresses the ACh-synthesizing enzyme choline acetyltransferase, the ACh-degrading enzyme acetylcholinesterase, two muscarinic ACh receptors (mAChRs), M3 and M4, and several nicotinic ACh receptors (nAChRs), including both α7- and α9-made homomeric nAChRs and predominantly the α3β2 ± α5 subtype of heteromeric nAChRs. Wounding affected the expression patterns of cholinergic molecules in the murine corneal epithelium. Constant stimulation of CECs through both muscarinic and nicotinic signaling pathways was essential for CEC survival and both directional and random migration in vitro. Both α7 and non-α7 nAChRs elicited chemotaxis, with the α7 signaling exhibiting a stronger chemotactic effect. Cholinergic stimulation of CECs upregulated expression of the integrin and cadherin molecules involved in epithelialization. We found synergy between the proepithelialization signals elicited by different ACh receptors expressed in CECs. Conclusions. Simultaneous stimulation of mAChRs and nAChRs by ACh may be required to synchronize and balance ionic and metabolic events in a single cell. Localization of these cholinergic enzymes and receptors in murine cornea indicated that the concentration of

  5. The effect of the corneal epithelium on the intraocular penetration of fluoroquinolone ophthalmic solution.

    PubMed

    Fukuda, Masamichi; Inoue, Amane; Sasaki, Kazuyuki; Takahashi, Nobuo

    2004-01-01

    Pharmacokinetic studies of antibacterial agents for infectious eye diseases have usually been performed on normal rabbit eyes. In this study, the intraocular penetration of fluoroquinolone ophthalmic solutions was determined in normal rabbit eyes and in rabbit eyes that had the corneal epithelium intentionally removed. We determined the intraocular penetration of ofloxacin (OFLX), levofloxacin (LVFX), and norfloxacin (NFLX), fluoroquinolone ophthalmic solutions that are already on the market and undergoing clinical studies, by injecting 50 microl of each solution into the cul-de-sacs of rabbit eyes three times at 15-min intervals. The drug concentration at 10, 30, 60, 120, and 240 min after final instillation was determined by high-performance liquid chromatography. The maximum concentration in the aqueous humor of normal rabbit eyes was 2.09 +/- 1.56 microg/ml (60 min, OFLX), 2.57 +/- 1.00 microg/ml (30 min, LVFX), and 0.42 +/- 0.12 microg/ml (120 min, NFLX). The drug concentration in the aqueous humor of eyes with intentionally removed corneal epithelium was 12.50 +/- 5.62 microg/ml (30 min, OFLX), 9.02 +/- 2.45 microg/ml (60 min, LVFX), and 8.54 +/- 5.17 microg/ml (30 min, NFLX). The drug penetration of the eye drops into eyes with removed corneal epithelium was around 6 times (OFLX), 3.5 times (LVFX), and 20 times (NFLX) higher than the penetration into the eye with normal cornea. Among the pharmacokinetic parameters of the three ophthalmic solutions according to the one-compartment model, the maximum concentration in the aqueous and the area under the concentration-time curve in the aqueous tended to be higher in the eyes with intentionally removed corneal epithelia than in those with normal corneas.

  6. Transepithelial Versus Epithelium-Off Corneal Collagen Cross-Linking for Progressive Keratoconus: A Prospective Randomized Controlled Trial.

    PubMed

    Al Fayez, Mashhoor F; Alfayez, Salman; Alfayez, Yasmin

    2015-10-01

    To compare the safety and efficacy of transepithelial with epithelium-off corneal cross-linking for progressive keratoconus. In a prospective clinical trial, 70 patients with progressive keratoconus were randomized to undergo corneal cross-linking with intact epithelium (n = 34) or after deepithelialization (n = 36). The main outcome measure was a change in the maximum K reading (K(max)). With 3-year follow-up, K(max) decreased in the epithelium-off group with a mean of 2.4 D and no patient showed evidence of progression. In the transepithelial group, K(max) increased by a mean of 1.1 D, and 20 patients (55%) showed progression of keratoconus. In this study, epithelium-off was significantly more effective than transepithelial corneal cross-linking in halting the progression of keratoconus (P < 0.0001).

  7. Epithelium-off versus transepithelial corneal collagen crosslinking for progressive corneal ectasia: a randomised and controlled trial.

    PubMed

    Rush, Sloan W; Rush, Ryan B

    2017-04-01

    To compare the outcomes of corneal collagen crosslinking (CXL) for the treatment of progressive corneal ectasia using a standard epithelium-off technique versus a transepithelial technique with enhanced riboflavin solution. One hundred and forty-four eyes with progressive corneal ectasia were prospectively randomised into a transepithelial CXL study arm or an epithelium-off CXL control arm. Follow-up examinations were set at 3, 6, 12 and 24 months. The primary outcome measure was change in the maximum simulated keratometry value (Ksteep) after 24 months of follow-up. The secondary outcome measure was change in the best spectacle-corrected visual acuity (BSCVA) after 24 months follow-up. One hundred and thirty-one eyes completed the 24-month follow-up interval. Change in Ksteep was -1.52±0.66 dioptres (D) for the control group versus -0.54±0.58 D for the study group at 24 months of follow-up (p=0.0320). Change in BSCVA was -0.18±0.09 logMAR for the control group versus -0.14±0.08 logMAR for the study group at 24 months of follow-up (p=0.4978). Two eyes in the control group had minor postoperative complications that did not affect the final visual acuity, and one eye in the control group underwent keratoplasty during the study interval. At 24 months of follow-up, subjects in the epithelium-off CXL group demonstrated a greater improvement in Ksteep compared with subjects in the transepithelial CXL group, but no statistically significant difference in BSCVA was found between groups. NCT01708538; pre-results. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  8. Morphological and autoradiographic studies on the corneal and limbal epithelium of rabbits.

    PubMed

    Góes, Rejane Maira; Barbosa, Flávia Leão; De Faria-E-Sousa, Sidney Júlio; Haddad, Antonio

    2008-02-01

    The investigation was centered on the morphological features of the conjunctiva-cornea transition (limbus) of the rabbit eye and the proliferative behavior of its epithelium. The eyes were processed for examination with light and electron microscopy, as well as for autoradiography after intravitreal injection of [(3)H]thymidine ([(3)H]TdR). At the sites of extraocular muscle insertion, the vascularization of the stroma extended to the peripheral cornea, and the limbal epithelium was thin with its basal stratum made up by clear cuboidal cells. In between the muscle insertions, the cuboidal clear cells, as well as the stroma blood vessels, were scarce. At the light microscope level, the basement membrane was distinct in the cornea but not in the limbus or the conjunctiva. Autoradiographs demonstrated that, at the limbus, the basal cells migrated very quickly to the suprabasal region and remained there up to the 28-day interval. Labeled cells were identified in all epithelial layers of the cornea, including the basal one, at 21 and 28 days but not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to that observed for the transition with scarce clear cells. The high-resolution autoradiographs demonstrated that the basal cuboidal clear limbal cells exhibit a quick renewal and that they are not label-retaining cells. These latter ones were detected all over the corneal epithelium and in the suprabasal layers of the limbus up to 28 days, in physiological conditions, without the need of stimulation by damage to the corneal epithelium. (c) 2008 Wiley-Liss, Inc.

  9. Aqueous extract of Centella asiatica promotes corneal epithelium wound healing in vitro.

    PubMed

    Ruszymah, Bt Hj Idrus; Chowdhury, Shiplu Roy; Manan, Nur Azeanty Bt Abdul; Fong, Ooi Sew; Adenan, Mohd Ilham; Saim, Aminuddin Bin

    2012-03-27

    Centella asiatica is a traditional herbal medicine that has been shown to have pharmacological effect on skin wound healing, and could be potential therapeutic agent for corneal epithelial wound healing. This study was done to evaluate the effects of Centella asiatica on the proliferation and migration of rabbit corneal epithelial (RCE) cells in the in vitro wound healing model. RCE cells were cultured with or without supplementation of Centella asiatica aqueous extract. Viability and proliferation of the RCE cells was determined by MTT assay and cell cycle was analyzed by flow cytometry. In vitro re-epithelization was studied by scratch assay and migration rate was evaluated quantitatively by image analyzer. Expression of corneal specific differentiation markers, CK12 and connexin 43, were studied via RT-PCR. It was found that supplementation of Centella asiatica did not show any significant effect on the RCE cells proliferation at the concentration up to 500ppm, while at the concentration of 1000ppm significantly inhibited RCE cells proliferation (p<0.05). However, at the concentration up to 62.5ppm, RCE cells shows significant enhancement of migration rate compared to the control group (p<0.05). It was also found that the supplementation of Centella asiatica aqueous extract did not alter the expression of differentiation markers and cell cycle. In conclusion, supplementation of Centella asiatica aqueous extract at low concentrations could be useful to promote corneal epithelium wound healing. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  10. In-vivo human corneal nerve imaging using Fourier-domain OCT

    NASA Astrophysics Data System (ADS)

    Shin, Jun Geun; Lee, Byeong Ha; Eom, Tae Joong; Hwang, Ho Sik

    2015-03-01

    We have imaged human corneal nerve bundles by using real-time Fourier-domain OCT (FD-OCT). Corneal nerves contribute to the maintenance of healthy ocular surface owing to their trophic influences on the corneal epithelium. The FD-OCT system was based on a swept laser of a 50 kHz sweeping rate and 1.31 μm center wavelength. At the area including sclera, limbus, and cornea, we could successfully get the in-vivo tomograms of the corneal nerve bundles. The scan range was 5 x 5mm. In this study, the A-scan images in each B-scan were realigned to have a flat air-surface boundary in the final B-scan image. With this effort, we could align corneal nerve bundle in a same depth and get the 3D image showing the branched and threadlike corneal nerve bundles.

  11. Substrates for Expansion of Corneal Endothelial Cells towards Bioengineering of Human Corneal Endothelium

    PubMed Central

    Navaratnam, Jesintha; Utheim, Tor P.; Rajasekhar, Vinagolu K.; Shahdadfar, Aboulghassem

    2015-01-01

    Corneal endothelium is a single layer of specialized cells that lines the posterior surface of cornea and maintains corneal hydration and corneal transparency essential for vision. Currently, transplantation is the only therapeutic option for diseases affecting the corneal endothelium. Transplantation of corneal endothelium, called endothelial keratoplasty, is widely used for corneal endothelial diseases. However, corneal transplantation is limited by global donor shortage. Therefore, there is a need to overcome the deficiency of sufficient donor corneal tissue. New approaches are being explored to engineer corneal tissues such that sufficient amount of corneal endothelium becomes available to offset the present shortage of functional cornea. Although human corneal endothelial cells have limited proliferative capacity in vivo, several laboratories have been successful in in vitro expansion of human corneal endothelial cells. Here we provide a comprehensive analysis of different substrates employed for in vitro cultivation of human corneal endothelial cells. Advances and emerging challenges with ex vivo cultured corneal endothelial layer for the ultimate goal of therapeutic replacement of dysfunctional corneal endothelium in humans with functional corneal endothelium are also presented. PMID:26378588

  12. Bioavailability of antioxidants applied to stratified human corneal epithelial cells.

    PubMed

    Stoddard, Alexander R; Koetje, Leah R; Mitchell, Anna K; Schotanus, Mark P; Ubels, John L

    2013-09-01

    Oxidative damage to the corneal epithelium may be involved in dry eye disease. The bioavailability and efficacy of antioxidants in human corneal limbal epithelial (HCLE) cells were measured to determine whether antioxidants might be beneficial constituents of lubricant eye drops. The activity of antioxidants was evaluated using a cellular antioxidant activity assay in which, cells were loaded with the reactive oxygen species (ROS)-sensitive fluorescent indicator, 2',7'-dichlorofluorescin diacetate (DCFH-DA), and an antioxidant compound. ROS were then generated intracellularly using 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) or extracellularly using xanthine oxidase, and the ability of an antioxidant to inhibit ROS-generated fluorescence was measured. When ROS were generated by ABAP, EC50 values for quercetin, epigallocatechin gallate (EGCG), n-propyl gallate, and gallic acid were 2.98, 3.41, 6.30, and 50.7 μM, respectively. When ROS were generated extracellularly by xanthine oxidase, EC50 values for quercetin, EGCG, n-propyl gallate, and gallic acid were 41.3, 56.5, 70.5, and 337.5 μM. These values were reduced significantly when an antioxidant was present both in the medium with the xanthine oxidase and within the cells. The antioxidants were effective at quenching ROS in HCLE cells, indicating that they are bioavailable and might be effective in protecting the corneal epithelium from oxidative damage if included in a lubricant eye drop.

  13. Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling

    SciTech Connect

    Seomun, Young; Joo, Choun-Ki

    2008-07-18

    Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins {alpha}2 and {beta}1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin {beta}1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

  14. Human tears reveal insights into corneal neovascularization.

    PubMed

    Zakaria, Nadia; Van Grasdorff, Sigi; Wouters, Kristien; Rozema, Jos; Koppen, Carina; Lion, Eva; Cools, Nathalie; Berneman, Zwi; Tassignon, Marie-José

    2012-01-01

    Corneal neovascularization results from the encroachment of blood vessels from the surrounding conjunctiva onto the normally avascular cornea. The aim of this study is to identify factors in human tears that are involved in development and/or maintenance of corneal neovascularization in humans. This could allow development of diagnostic tools for monitoring corneal neovascularization and combination monoclonal antibody therapies for its treatment. In an observational case-control study we enrolled a total of 12 patients with corneal neovascularization and 10 healthy volunteers. Basal tears along with reflex tears from the inferior fornix, superior fornix and using a corneal bath were collected along with blood serum samples. From all patients, ocular surface photographs were taken. Concentrations of the pro-angiogenic cytokines interleukin (IL)-6, IL-8, Vascular Endothelial Growth Factor (VEGF), Monocyte Chemoattractant Protein 1 (MCP-1) and Fas Ligand (FasL) were determined in blood and tear samples using a flow cytometric multiplex assay. Our results show that the concentration of pro-angiogenic cytokines in human tears are significantly higher compared to their concentrations in serum, with highest levels found in basal tears. Interestingly, we could detect a significantly higher concentration of IL- 6, IL-8 and VEGF in localized corneal tears of patients with neovascularized corneas when compared to the control group. This is the first study of its kind demonstrating a significant difference of defined factors in tears from patients with neovascularized corneas as compared to healthy controls. These results provide the basis for future research using animal models to further substantiate the role of these cytokines in the establishment and maintenance of corneal neovascularization.

  15. Human Tears Reveal Insights into Corneal Neovascularization

    PubMed Central

    Wouters, Kristien; Rozema, Jos; Koppen, Carina; Lion, Eva; Cools, Nathalie; Berneman, Zwi; Tassignon, Marie-José

    2012-01-01

    Corneal neovascularization results from the encroachment of blood vessels from the surrounding conjunctiva onto the normally avascular cornea. The aim of this study is to identify factors in human tears that are involved in development and/or maintenance of corneal neovascularization in humans. This could allow development of diagnostic tools for monitoring corneal neovascularization and combination monoclonal antibody therapies for its treatment. In an observational case-control study we enrolled a total of 12 patients with corneal neovascularization and 10 healthy volunteers. Basal tears along with reflex tears from the inferior fornix, superior fornix and using a corneal bath were collected along with blood serum samples. From all patients, ocular surface photographs were taken. Concentrations of the pro-angiogenic cytokines interleukin (IL)-6, IL-8, Vascular Endothelial Growth Factor (VEGF), Monocyte Chemoattractant Protein 1 (MCP-1) and Fas Ligand (FasL) were determined in blood and tear samples using a flow cytometric multiplex assay. Our results show that the concentration of pro-angiogenic cytokines in human tears are significantly higher compared to their concentrations in serum, with highest levels found in basal tears. Interestingly, we could detect a significantly higher concentration of IL- 6, IL-8 and VEGF in localized corneal tears of patients with neovascularized corneas when compared to the control group. This is the first study of its kind demonstrating a significant difference of defined factors in tears from patients with neovascularized corneas as compared to healthy controls. These results provide the basis for future research using animal models to further substantiate the role of these cytokines in the establishment and maintenance of corneal neovascularization. PMID:22590547

  16. Cultured human ocular surface epithelium on therapeutic contact lenses

    PubMed Central

    Girolamo, Nick Di; Chui, Jeanie; Wakefield, Denis; Coroneo, Minas T

    2007-01-01

    Background This study was initiated after observation of some intriguing epithelial growth properties of contact lenses used as a bandage for patients after pterygium surgery. Aim To determine the efficacy of culturing human ocular surface epithelial cells on therapeutic contact lenses in autologous serum with a view of using this system to transfer epithelial cells to patients with persistent corneal or limbal defects. Methods Excess graft tissue resected from patients undergoing pterygium surgery (n = 3) consisting of limbal epithelium was placed on siloxane–hydrogel contact lenses (lotrafilcon A and balafilcon A). Limbal explants were cultured in media with 10% autologous serum. Morphology, proliferative capacity and cytokeratin profile were determined by phase contrast, light and electron microscopy, and immunohistochemical analysis. Results Lotrafilcon A contact lenses sustained proliferation and migration from limbal tissue. Cells became confluent after 10–14 days and consisted of 2–3 layers with a corneal phenotype (CK3+/CK12+/CK19−) and a propensity to proliferate (p63+). Electron microscopy showed microvilli on the apical surface with adhesive projections, indicating that these cells were stable and likely to survive for a long term. Growth was not observed from limbal explants cultured on balafilcon A contact lenses. Conclusion A method for culturing human ocular surface epithelium on contact lenses that may facilitate expansion and transfer of autologous limbal epithelial cells while avoiding the risks associated with transplanting allogeneic tissue has been developed. This technique may be potentially useful for the treatment of patients with limbal stem cell deficiency. PMID:16987897

  17. Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity

    PubMed Central

    Shetty, Rohit; Sathyanarayanamoorthy, Arunapriya; Ramachandra, Reshma Airody; Arora, Vishal; Ghosh, Anuprita; Srivatsa, Purnima Raman; Pahuja, Natasha; Nuijts, Rudy M. M. A.; Sinha-Roy, Abhijit; Ghosh, Arkasubhra

    2015-01-01

    Purpose Keratoconus (KC) is characterized by progressive vision loss due to corneal thinning and structural abnormalities. It is hypothesized that KC is caused by deregulated collagen levels and collagen fibril-maturating enzyme lysyl oxidase (LOX). Further, it is currently not understood whether the gene expression deregulated by the corneal epithelium influences KC pathogenesis. We studied (i) the expressions of the LOX, collagen I (COL IA1), collagen IV (COL IVA1), MMP9, and IL6 genes in KC corneal epithelia, (ii) validated their expression levels in patient tissues, and (iii) correlated expression levels with KC disease severity. The primary goal of this study was to evaluate the importance of these genes in the progression of KC. Methods We analyzed the gene expression levels of the key proteins LOX, collagens (COL IA1 and COL IVA1), MMP9, and IL6 in debrided corneal epithelia from a large cohort of KC patients (90 eyes) and compared them to control patients (52 eyes) without KC. We measured the total LOX activity in the tears of KC patients compared to controls. We also correlated the protein expression levels of LOX and collagens by immunohistochemistry (IHC) in primary tissues from KC patients (27 eyes) undergoing keratoplasty compared to healthy donor corneas (15 eyes). Results We observed a significant reduction in LOX transcript levels in KC corneal epithelia, and LOX activity in KC tears correlated with disease severity. Collagen transcripts were also reduced in KC while MMP9 transcript levels were upregulated and correlated with disease severity. IL6 was moderately increased in KC patients. IHC demonstrated a reduction in the protein expression levels of LOX in the epithelium and collagen IV in the basement membrane of KC patients compared to healthy donor corneas. Conclusions The data demonstrates that the structural deformity of the KC cornea may be dependent on reduced expressions of collagens and LOX, as well as on MMP9 elevated by the corneal

  18. In vitro eye irritancy test of lauryl derivatives using the reconstructed rabbit corneal epithelium model.

    PubMed

    Matsuda, Sanae; Hisama, Masayoshi; Shibayama, Hiroharu; Itou, Norihiko; Iwaki, Masahiro

    2009-06-01

    The rabbit corneal epithelium model (RCE model) was developed as a three-dimensional in vitro model to replace animal testing for the assessment of eye tolerance. In the model, a stratified culture of rabbit corneal epithelial cells is grown at the air-liquid interface on an amniotic membrane acting as a parabasal membrane. The alkaline exposure was restored each day in the presence of no irritants, although with the addition of SLS, which is a major irritant, the restoration of deficit was inhibited on the RCE model in a dose-dependent manner. The results of this test were comparable with those of the Draize test, and thus, this method using the RCE model may prove to be a useful and sensitive in vitro eye irritation test. The lauryl fatty chain derivatives, such as polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-l-glutaminate (SLG), which are widely used as surfactants for toiletry products and cosmetics, were evaluated for in vitro eye irritation potential using the RCE model. SLS, PLE, SPLE, MGL, and SLG inhibited 88.7%, 59.2%, 69.0%, 47.5%, and 15.7% of the restoration of deletion 24h after treatment at a concentration of 0.05%. The IC(50) (50% inhibitory concentration) values of SLS, PLE, SPLE, MGL, and SLG were 0.002%, 0.021%, 0.005%, 0.056%, and 0.448%, respectively. These results indicated that a functional group at the end of lauryl chain is an important factor for inhibiting the restoration of deletion using the RCE model.

  19. Involvement of pigment epithelium-derived factor, docosahexaenoic acid and neuroprotectin D1 in corneal inflammation and nerve integrity after refractive surgery.

    PubMed

    Kenchegowda, S; He, J; Bazan, H E P

    2013-01-01

    Alterations in corneal innervations result in impaired corneal sensation, severe dry eye and damage to the epithelium that may in turn lead to corneal ulcers, melting and perforation. These alterations can occur after refractive surgery. We have discovered that pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA or the docosanoid bioactive neuroprotectin D1 (NPD1)) induces nerve regeneration after corneal surgery that damages the stromal nerves. We found that PEDF is released from corneal epithelial cells after injury, and when DHA is provided to the cells it stimulates the biosynthesis of NPD1 by an autocrine mechanism. The combination of PEDF plus DHA also decreased the production of leukotriene B4 (LTB4), a neutrophil chemotactic factor, thereby decreasing the inflammation induced after corneal damage. These studies suggest that PEDF plus DHA and its derivative NPD1 hold promise as a future treatment to restore a healthy cornea after nerve damage. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Effects of fluoroquinolone eye solutions without preservatives on human corneal epithelial cells in vitro.

    PubMed

    Oum, Boo Sup; Kim, Na Mi; Lee, Jong Soo; Park, Young Min

    2014-01-01

    To evaluate the biologic effects of fluoroquinolone eye solutions without preservatives on cultured human corneal epithelial cells in vitro. We studied the effect of diverse generations of topical fluoroquinolones such as ofloxacin 0.3%, levofloxacin 0.5%, tosufloxacin 0.3%, moxifloxacin 0.5% and gatifloxacin 0.3% on cultured human corneal epithelial cells. MTT-based calorimetric assay, lactate dehydrogenase leakage (LDH) assay and scratch wound test were performed. Corneal epithelial cell morphologies were examined by performing inverted light microscopy and transmission electron microscopy. In all topical fluoroquinolones, the metabolic activity of the corneal epithelial cells decreased in a time-dependent fashion and the LDH titer increased with longer exposure times. Especially, the LDH titers significantly increased after exposure to moxifloxacin 0.5% and gatifloxacin 0.3% compared with controls. The migration rates of the corneal epithelial cells were faster in ofloxacin 0.3% or levofloxacin 0.5% than other fluoroquinolones. Severe cellular morphological damage was observed after exposure to moxifloxacin 0.5% and gatifloxacin 0.3%. As moxifloxacin 0.5% and gatifloxacin 0.3% induced the toxic effect to the corneal epithelial cells, compared with other fluoroquinolones, the 4th fluoroquinolone eye solutions should be carefully used in case of the corneal epithelium is damaged by long duration of treatment or overdosage. © 2014 S. Karger AG, Basel.

  1. Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system

    PubMed Central

    Liang, Hong; Pauly, Aude; Riancho, Luisa; Baudouin, Christophe

    2011-01-01

    Aims Using an established three-dimensional (3D) toxicological model based on reconstituted human corneal epithelium (HCE), this study investigated the tolerability of four topical intraocular-pressure-lowering agents: the commercial solutions of benzalkonium chloride (BAC)-containing 0.005% latanoprost, 0.004% travoprost, 0.03% bimatoprost containing 0.02%, 0.015% and 0.005% BAC, respectively, and the preservative-free (PF) tafluprost. Solutions of 0.01% and 0.02% BAC alone were also evaluated for comparison. Methods The 3D-HCEs were treated with solutions for 24 h followed or not by a 24 h recovery period. We used a modified MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) procedure to assess cell viability in the HCE. Frozen sections of HCE were analysed using fluorescence microscopy for the evaluation of apoptosis (terminal deoxynucleotidyl transferase mediated dUTP nick end labelling), inflammation (ICAM-1) and proliferation (Ki67). Corneal epithelial tight junctions (occludin and tight junction protein 1 (zona occludens 1)) were also assessed by en face confocal microscopy in response to the different eye-drops. Results The MTT test revealed that the cytotoxicity of antiglaucoma eye-drops was primarily related to the concentration of their common BAC preservative (0.02% BAC-latanoprost>0.015% BAC-travoprost>0.005% BAC-bimatoprost). PF-tafluprost did not induce any obvious cytotoxicity, showed the least expression of inflammatory or apoptotic markers and revealed preservation of membrane immunostaining of tight junction proteins in comparison with BAC-containing solutions. Conclusion The toxicological model of the 3D reconstructed corneal epithelia model confirmed the ocular surface cytotoxicity of BAC-containing antiglaucomatous solutions. Compared with the formulations containing the toxic preservative BAC, PF-tafluprost was well tolerated without inducing significant corneal epithelium deterioration. PMID:21429894

  2. Therapeutic efficiency of tissue-engineered human corneal endothelium transplants on rabbit primary corneal endotheliopathy.

    PubMed

    Fan, Ting-jun; Zhao, Jun; Hu, Xiu-zhong; Ma, Xi-ya; Zhang, Wen-bo; Yang, Chao-zhong

    2011-06-01

    To evaluate the therapeutic efficiency of tissue-engineered human corneal endothelia (TE-HCEs) on rabbit primary corneal endotheliopathy (PCEP), TE-HCEs reconstructed with monoclonal human corneal endothelial cells (mcHCECs) and modified denuded amniotic membranes (mdAMs) were transplanted into PCEP models of New Zealand white rabbits using penetrating keratoplasty. The TE-HCEs were examined using diverse techniques including slit-lamp biomicroscopy observation and pachymeter and tonometer measurements in vivo, and fluorescent microscopy, alizarin red staining, paraffin sectioning, scanning and transmission electron microscopy observations in vitro. The corneas of transplanted eyes maintained transparency for as long as 200 d without obvious edema or immune rejection. The corneal thickness of transplanted eyes decreased gradually after transplanting, reaching almost the thickness of normal eyes after 156 d, while the TE-HCE non-transplanted eyes were turbid and showed obvious corneal edema. The polygonal corneal endothelial cells in the transplanted area originated from the TE-HCE transplant. An intact monolayer corneal endothelium had been reconstructed with the morphology, cell density and structure similar to those of normal rabbit corneal endothelium. In conclusion, the transplanted TE-HCE can reconstruct the integrality of corneal endothelium and restore corneal transparency and thickness in PCEP rabbits. The TE-HCE functions normally as an endothelial barrier and pump and promises to be an equivalent of HCE for clinical therapy of human PCEP.

  3. Tolerance of human corneal endothelium to glycerol.

    PubMed

    Brunette, I; Nelson, L R; Bourne, W M

    1989-12-01

    As an initial step in the development of a method for corneal cryopreservation by vitrification, we attempted to establish the maximum concentration of glycerol to which human corneal endothelium could be exposed at 4 degrees C for 15 min without damage. Damage was defined as an increase in mean endothelial cell size or the inability to maintain corneal thickness for 1 week after exposure to glycerol. Using a system for long-term corneal perfusion, we perfused 24 paired human corneas with glycerol at 4 degrees C. The concentration of glycerol increased at a rate of 20% (w/v) (2.2 M) per hour until the desired maximum concentration was reached for that cornea, stabilized for 15 min, and then decreased at the same rate. The corneas were then perfused at 37 degrees C with Dulbecco's medium at a rate of 5 microliters/min under 18 mm Hg intracameral pressure for 7 days with daily measurements of corneal thickness. Endothelial morphology was examined by specular microscopy and by scanning electron microscopy. After 7 days of perfusion at 37 degrees C, there was a statistically significant direct relationship between the maximum concentration of glycerol to which the experimental eyes had been exposed and the increase in mean endothelial cell size. The mean endothelial cell size increased in corneas exposed to glycerol concentrations of 40, 50, and 60% (w/v), but did not differ significantly from baseline measurements in the corneas exposed to 30% glycerol or less. Thus, there was no detectable damage to human corneas exposed to 30% (w/v) (3.3 M) glycerol in this system. Tolerance of higher concentrations may be achieved by changes in the rates of addition and removal of glycerol or in the composition of the perfusate.

  4. Epithelium

    MedlinePlus

    The term "epithelium" refers to layers of cells that line hollow organs and glands. It is also those cells that make ... Epithelium. In: Kierszenbaum AL, Tres LL. Histology and Cell Biology - An Introduction to Pathology , 3rd ed. Philadelphia, ...

  5. Human excimer laser corneal surgery: preliminary report.

    PubMed Central

    L'Esperance, F A; Taylor, D M; Del Pero, R A; Roberts, A; Gigstad, J; Stokes, M T; Warner, J W; Telfair, W B; Martin, C A; Yoder, P R

    1988-01-01

    The first human trial utilizing the argon fluoride excimer laser at 193 nm to produce a superficial keratectomy in ten human eyes has been described with the histopathological evaluation of four eyes and the longer gross appearance of six eyes at intervals extending to 10 months post-excimer laser treatment. The process of laser superficial keratectomy has proved to be one of the promising areas of surgical intervention for reconstructive or refractive keratoplasty in the future. Intensive investigations need to be undertaken on the corneal wound healing process following laser ablation as well as the nature, and long-term stability of the corneal excisions or induced refractive corrections. It is essential that the optimal laser parameters be established for the various refractive corrections and other corneal surgical techniques, and that pathophysiologic and histopathologic changes that have been induced by the excimer laser-corneal tissue interaction in animals and humans be critically and extensively analyzed. Images FIGURE 1 FIGURE 19 A FIGURE 19 B FIGURE 20 A FIGURE 20 B FIGURE 21 A FIGURE 21 B FIGURE 22 A FIGURE 22 B FIGURE 23 FIGURE 24 FIGURE 25 FIGURE 26 FIGURE 27 FIGURE 28 FIGURE 29 A FIGURE 29 B FIGURE 29 C FIGURE 29 D FIGURE 30 A FIGURE 30 B FIGURE 31 A FIGURE 31 B FIGURE 32 FIGURE 33 FIGURE 34 FIGURE 35 FIGURE 36 FIGURE 37 A FIGURE 37 B FIGURE 37 C FIGURE 38 A FIGURE 38 B FIGURE 39 A FIGURE 39 B FIGURE 39 C FIGURE 40 A FIGURE 40 B PMID:2979049

  6. Proteomic features of delayed ocular symptoms caused by exposure to sulfur mustard: As studied by protein profiling of corneal epithelium.

    PubMed

    Pajoohesh, Maryam; Naderi, Mostafa; Naderi-Manesh, Hossein

    2017-11-01

    Exposure to mustard gas can lead to variations in the proteome of corneal epithelium cells and after a latency period produces delayed symptoms in the eyes of chemical victims. Hence, a comparative proteome analysis was conducted between the corneal epithelial cells of chemical victims from Iran-Iraq war (1980-1988) and healthy donors. To this end, corneal epithelium samples from victims and healthy individuals were collected, and the proteome of these samples were prepared for two-dimensional electrophoresis and the analysis of spots by statistical software. Selected spots were further analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Twenty four proteins were identified of which eighteen proteins showed downregulation while six proteins were upregulated in the victims in comparison to the normal individuals. Also, six protein spots were confirmed by western-blot analysis. In conclusion, all the twenty-four identified proteins are involved in pathways which their up- or down-regulation leads to the accumulation of undesired substrates, cell death and apoptosis. Bioinformatics' tools indicated that these identified proteins were involved in various metabolic processes, DNA damage response, immune response and etc. The present study provides a suitable platform for further clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. In Vivo Confocal Microscopic Changes of the Corneal Epithelium and Stroma in Patients With Herpes Zoster Ophthalmicus

    PubMed Central

    Hamrah, Pedram; Sahin, Afsun; Dastjerdi, Mohammad H.; Shahatit, Bashar M.; Bayhan, Hasan A.; Dana, Reza; Pavan-Langston, Deborah

    2016-01-01

    Purpose To analyze the density and morphology of corneal epithelial cells and keratocytes by in vivo confocal microscopy (IVCM) in patients with herpes zoster ophthalmicus (HZO) as associated with corneal innervation. Design Prospective, controlled and masked cross-sectional study. Methods SETTING: Single-center study. PATIENTS: Thirty eyes with the diagnosis HZO and their contralateral clinically unaffected eyes, 15 eyes of 15 normal controls. INTERVENTION PROCEDURES: In vivo confocal microscopy and corneal esthesiometry of the central cornea. MAIN OUTCOME MEASURES: Changes in morphology and density of the superficial and basal epithelial cells and stromal keratocytes, and correlation with corneal sensation. Results The density of superficial epithelial cells in HZO eyes with severe sensation loss (766.5 ± 25.2 cells/mm2) was significantly lower than both healthy control eyes (1450.23 ± 150.83 cells/mm2) and contralateral unaffected eyes (1974.13 ± 298.24 cells/mm2) (P = .003). Superficial epithelial cell size (1162.5 μm2) was significantly larger in HZO eyes with severe loss of sensation, as compared to contralateral (441.46 ± 298.14) or healthy eyes (407.4 ± 47.2mm2; all P < .05). The density of basal epithelial cells, anterior keratocytes, and posterior keratocytes did not show statistical significance between patients, controls, and contralateral unaffected eyes. Changes in superficial epithelial cell density and morphology correlated strongly with corneal sensation. Conclusions In vivo confocal microscopy reveals profound HZO-induced changes in the superficial epithelium, as demonstrated by increase in cell size, decrease in cell density, and squamous metaplasia. We demonstrate that these changes strongly correlate with changes in corneal innervation in eyes affected by HZO. PMID:25748579

  8. Reciprocal transmission of herpes simplex virus type 1 (HSV-1) between corneal epithelium and trigeminal neurites in an embryonic chick organ culture.

    PubMed

    Hafezi, Wali; Eing, Bodo R; Lorentzen, Eva U; Thanos, Solon; Kühn, Joachim E

    2002-06-01

    Reciprocal transmission between epithelia and sensory neurons of the peripheral nervous system is a crucial step in the life cycle of herpes simplex virus type 1 (HSV-1) and related alphaherpesviruses. In searching for an easy-to-perform and generally applicable experimental approach that enables the direct analysis of virus transfer between primary epithelial cells and sensory neurites, we investigated the spread of HSV-1 in a dual-chamber organ model comprising chick embryonic corneal epithelia and trigeminal sensory neurons. Embryonic chick corneal and trigeminal tissues were found to be permissive for productive infection with HSV-1. Our data show that HSV-1 efficiently enters neurites re-innervating the cornea and reaches the ganglion explant by retrograde axonal transport, with the first antigen-positive cells being detectable approximately 12 h postinfection. After direct infection of trigeminal tissues, the virus is transported by anterograde axonal transport to the corneal epithelium, causing a visible cytopathic effect approximately 48 h postinfection. These results suggest that the organ model presented in this study holds particular promise for the direct observation and molecular analysis of herpes simplex virus spread between primary epithelia and sensory neurons and that it may be an attractive alternative to current experimental approaches based on laboratory animals or human fetal tissues.

  9. Biomechanical and Histopathologic Effects of Pulsed-Light Accelerated Epithelium-On/-Off Corneal Collagen Cross-Linking.

    PubMed

    Zhang, Xiaoyu; Sun, Ling; Shen, Yang; Tian, Mi; Zhao, Jing; Zhao, Yu; Li, Meiyan; Zhou, Xingtao

    2017-07-01

    This study aimed to compare the biomechanical and histopathologic effects of transepithelial and accelerated epithelium-off pulsed-light accelerated corneal collagen cross-linking (CXL). A total of 24 New Zealand rabbits were analyzed after sham operation (control) or transepithelial or epithelium-off operation (45 mW/cm for both). The transepithelial group was treated with pulsed-light ultraviolet A for 5 minutes 20 seconds, and the epithelium-off group was treated for 90 seconds. Biomechanical testing, including ultimate stress, Young modulus, and the physiological modulus, was analyzed. Histological changes were evaluated by light microscopy and transmission electron microscopy. The stress-strain curve was nonlinear in both accelerated transepithelial and epithelium-off CXL groups. The stress and elastic moduli were all significantly higher in both experimental groups compared with the control group (P < 0.05), whereas there were no significant differences between the 2 treatment groups (P > 0.05). Six months after the operation, hematoxylin and eosin staining and transmission electron microscopy showed that the subcutaneous collagen fibers were arranged in a regular pattern, and the fiber density was higher in the experimental groups. Both transepithelial and accelerated epithelium-off CXL produced biomechanical and histopathologic improvements, which were not significantly different between the 2 pulsed-light accelerated CXL treatments.

  10. Insulin Restores an Altered Corneal Epithelium Circadian Rhythm in Mice with Streptozotocin-induced Type 1 Diabetes

    PubMed Central

    Song, Fang; Xue, Yunxia; Dong, Dong; Liu, Jun; Fu, Ting; Xiao, Chengju; Wang, Hanqing; Lin, Cuipei; Liu, Peng; Zhong, Jiajun; Yang, Yabing; Wang, Zhaorui; Pan, Hongwei; Chen, Jiansu; Li, Yangqiu; Cai, Dongqing; Li, Zhijie

    2016-01-01

    The mechanisms of corneal epithelial lesions and delayed wound repair, as well as their association with diabetes mellitus, are critical issues for clinical ophthalmologists. To test whether the diabetic condition alters the circadian rhythm in a mouse cornea and whether insulin can synchronise the corneal clock, we studied the effects of streptozotocin-induced diabetes on the mitosis of epithelial cells, the recruitment of leukocytes to the cornea, and the expression of main core clock genes (Clock, Bmal1, Per2, Cry1, and Rev-erbα) in the corneal epithelium. We also assessed the possible effect of insulin on these modifications. Diabetes downregulated Clock, Bmal1, and Per2 expression, upregulated Cry1 and Rev-erbα expression, reduced corneal epithelial mitosis, and increased leukocyte (neutrophils and γδ T-cells) recruitment to the cornea. Early treatments with insulin partially restored the altered rhythmicity in the diabetic cornea. In conclusion, insulin-dependent diabetes altered the normal rhythmicity of the cornea, and insulin administration had a beneficial effect on restoring normal rhythmicity in the diabetic cornea. PMID:27611469

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

    PubMed

    Haddad, A; Faria-e-Sousa, S J

    2014-06-01

    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.

  12. Proliferation of the vascular endothelium of the iris following total debridement of the corneal epithelium and limbal excision of rabbits.

    PubMed

    Góes, Rejane Maira; Barbosa, Flávia Leão; de Faria-E-Sousa, Sidney Júlio; Kajiwara, João Kazuyuki; Haddad, Antonio

    2008-07-01

    Damage to the corneal epithelium causes not only a reaction for its repair but also affects other parts of the cornea as well as different components of the anterior segment of the eye. The purpose of this investigation was to analyze the consequences, following epithelial and limbal damage, to the iris of rabbits (Oryctolagus cuniculus). The corneal epithelium was thoroughly scraped followed by surgical excision of the limbus. Next, (3)H-thymidine ((3)H-TdR) was injected intravitreally both into the right (experimental) and left (control) eyes which had their anterior segments processed for autoradiography at intervals of 2, 7 and 21 days after surgery (three rabbits per interval). The irises were also examined with scanning-electron and confocal microscopy after Evans blue injection. There was a high frequency of labeling in the cells of the iris blood vessels in the experimental eye, particularly the endothelial ones. The ratio of labeled cells between experimental and control irises was 40:1, with a population of nuclei increasing by 25% and remaining labeled up to 21 days. There was also an increase in the volume of the iris vasculature as shown by confocal microscopy. The high labeling frequencies of the vascular cells were observed throughout the iris from the ciliary to the pupillary regions. The lesions on the corneal epithelium elicit proliferation of the iris vascular cells, mainly its endothelium, as well as an early breakdown of the blood-aqueous barrier. The daughter cells resulting from the damage to the eye surface were detected up to 21 days after a single injection of (3)H-TdR, most likely due to their slow turnover. As a consequence of this proliferation, the vasculature of the iris increased in volume.

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

    PubMed Central

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

    2008-01-01

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

  14. Toxicity of Topical Antifungal Agents to Stratified Human Cultivated Corneal Epithelial Sheets

    PubMed Central

    Kimakura, Mikiko; Yokoo, Seiichi; Nakagawa, Suguru; Yamagami, Satoru; Amano, Shiro

    2014-01-01

    Abstract Purpose: Prolonged use of topical antifungal agents may compromise corneal epithelial integrity. Here, we used an in vitro model of human stratified corneal epithelium to compare the ocular toxicity profiles of 4 different antifungal eye drops. Methods: Human corneal epithelial cell sheets were cultured in a serum-free medium containing 0.1% micafungin, 1% voriconazole, 5% pimaricin, 0.1% amphotericin B, or controls (saline or 5% glucose). Cell viability and barrier function were measured by WST-1 assay and carboxyfluorescein permeability assay, respectively. Cell migration was measured on a wound healing assay. Results: WST-1 assay and carboxyfluorescein permeability assay revealed that amphotericin B was the most toxic drug, followed by pimaricin, micafungin, and voriconazole. Cell migration on a wound healing assay was decreased in the following order, amphotericin B, pimaricin, micafungin, and voriconazole. Conclusions: Topical micafungin and voriconazole appeared to be the least toxic to the corneal epithelium. Drug prescription should consider not only fungal species and susceptibility but also ocular toxicity and stage of treatment. PMID:25280055

  15. Activation of cell division and nucleic acid synthesis in the corneal epithelium of albino rats by repeated stress

    SciTech Connect

    Berezhnova, N.I.; Timoshin, S.S.

    1985-05-01

    Adaption to unfavorable factors is accompanied by activation of nucleic acid and protein synthesis in systems responsible for adaption. The authors investigate the possibility of similar changes taking place in structures not actively participating in adaptation. The corneas of the dead male albin rats were preincubated with tritium-uridine for 1.5 hours. The mitotic index, the index of tritium-thymidine-labeled nuclei and the intensity of thymidine labeling were determined. The results indicate that after a single exposure to hypoxia, hyperthermia, and immobilization, mitotic index in the corneal epithelium decreased and DNA synthesis under these circumstances remained stable.

  16. Human Growth Hormone Promotes Corneal Epithelial Cell Migration in Vitro

    PubMed Central

    Ding, Juan; Wirostko, Barbara; Sullivan, David A

    2015-01-01

    Purpose Corneal wound healing is a highly regulated process that requires the proliferation and migration of epithelial cells and interactions between epithelial cells and stromal fibroblasts. Compounds that can be applied topically to the ocular surface and that have the capability of activating corneal epithelial cells to proliferate and/or migrate would be useful to promote corneal wound healing. We hypothesize that human growth hormone (HGH) will activate Signal Transducer and Activators of Transcription-5 (STAT5) signaling and promote corneal wound healing by enhancing corneal epithelial cell and fibroblast proliferation and/or migration in vitro. The purpose of this study is to test these hypotheses. Methods We studied cell signaling, proliferation and migration using an immortalized human corneal epithelial cell line and primary human corneal fibroblasts in vitro. We also examined whether insulin-like growth factor-1 (IGF-1), a hormone known to mediate many of HGH’s growth promoting actions, may play a role in this effect. Results We show that HGH activates STAT5 signaling and promotes corneal epithelial cell migration in vitro. The migratory effect requires an intact communication between corneal epithelia and fibroblasts, and is not mediated by IGF-1. Conclusion HGH may represent a topical therapeutic to promote corneal epithelial wound healing. This warrants further investigation. PMID:25782399

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

    PubMed

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

    2016-05-01

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

  18. Coefficient of Friction of Human Corneal Tissue.

    PubMed

    Wilson, Tawnya; Aeschlimann, Rudolf; Tosatti, Samuele; Toubouti, Youssef; Kakkassery, Joseph; Osborn Lorenz, Katherine

    2015-09-01

    A novel property evaluation methodology was used to determine the elusive value for the human corneal coefficient of friction (CoF). Using a microtribometer on 28 fresh human donor corneas with intact epithelia, the CoF was determined in 4 test solutions (≥5 corneas/solution): tear-mimicking solution (TMS) in borate-buffered saline (TMS-PS), TMS in phosphate-buffered saline (TMS-PBS), TMS with HEPES-buffered saline (TMS-HEPES), and tear-like fluid in PBS (TLF-PBS). Mean (SD) CoF values ranged from 0.006 to 0.015 and were 0.013 (0.010) in TMS-PS, 0.006 (0.003) in TMS-PBS, 0.014 (0.005) in TMS-HEPES, and 0.015 (0.009) in TLF-PBS. Statistically significant differences were shown for TMS-PBS versus TLF (P = 0.0424) and TMS-PBS versus TMS-HEPES (P = 0.0179), but not for TMS-PBS versus TMS-PS (P = 0.2389). Successful measurement of the fresh human corneal tissue CoF was demonstrated, with values differing in the evaluated buffer solutions, within this limited sample size.

  19. Revisiting the human seminiferous epithelium cycle.

    PubMed

    Nihi, F; Gomes, M L M; Carvalho, F A R; Reis, A B; Martello, R; Melo, R C N; Almeida, F R C L; Chiarini-Garcia, H

    2017-06-01

    Can all types of testicular germ cells be accurately identified by microscopy techniques and unambiguously distributed in stages of the human seminiferous epithelium cycle (SEC)? By using a high-resolution light microscopy (HRLM) method, which enables an improved visualization of germ cell morphological features, we identified all testicular germ cells in the seminiferous epithelium and precisely grouped them in six well-delimitated SEC stages, thus providing a reliable reference source for staging in man. Morphological characterization of germ cells in human has been done decades ago with the use of conventional histological methods (formaldehyde-based fixative -Zenker-formal- and paraffin embedding). These early studies proposed a classification of the SEC in six stages. However, the use of stages as baseline for morphofunctional evaluations of testicular parenchyma has been difficult because of incomplete morphological identification of germ cells and their random distribution in the human SEC. Testicular tissue from adult and elderly donors with normal spermatogenesis according to Levin's, Johnsen's and Bergmann's scores were used to evaluate germ cell morphology and validate their distribution and frequency in stages throughout human spermatogenesis. Testicular tissue from patients diagnosed with congenital bilateral agenesis of vas deferens (n = 3 adults) or prostate cancer (n = 3 elderly) were fixed in glutaraldehyde and embedded in araldite epoxy resin. Morphological analyses were performed by both light and transmission electron microscopy. HRLM method enabled a reliable morphological identification of all germ cells (spermatogonia, spermatocytes and spermatids) based on high-resolution aspects of euchromatin, heterochromatin and nucleolus. Moreover, acrosomal development of spermatids was clearly revealed. Altogether, our data redefined the limits of each stage leading to a more reliable determination of the SEC in man. Occasionally, germ cells can be

  20. Human induced pluripotent stem cell differentiation and direct transdifferentiation into corneal epithelial-like cells

    PubMed Central

    Cieślar-Pobuda, Artur; Rafat, Mehrdad; Knoflach, Viktoria; Skonieczna, Magdalena; Hudecki, Andrzej; Małecki, Andrzej; Urasińska, Elżbieta; Ghavami, Seaid; Łos, Marek J.

    2016-01-01

    The corneal epithelium is maintained by a small pool of tissue stem cells located at the limbus. Through certain injuries or diseases this pool of stem cells may get depleted. This leads to visual impairment. Standard treatment options include autologous or allogeneic limbal stem cell (LSC) transplantation, however graft rejection and chronic inflammation lowers the success rate over long time. Induced pluripotent stem (iPS) cells have opened new possibilities for treating various diseases with patient specific cells, eliminating the risk of immune rejection. In recent years, several protocols have been developed, aimed at the differentiation of iPS cells into the corneal epithelial lineage by mimicking the environmental niche of limbal stem cells. However, the risk of teratoma formation associated with the use of iPS cells hinders most applications from lab into clinics. Here we show that the differentiation of iPS cells into corneal epithelial cells results in the expression of corneal epithelial markers showing a successful differentiation, but the process is long and the level of gene expression for the pluripotency markers does not vanish completely. Therefore we set out to determine a direct transdifferentiation approach to circumvent the intermediate state of pluripotency (iPS-stage). The resulting cells, obtained by direct transdifferentiation of fibroblasts into limbal cells, exhibited corneal epithelial cell morphology and expressed corneal epithelial markers. Hence we shows for the first time a direct transdifferentiation of human dermal fibroblasts into the corneal epithelial lineage that may serve as source for corneal epithelial cells for transplantation approaches. PMID:27275539

  1. Potential role for laminin 5 in hypoxia-mediated apoptosis of human corneal epithelial cells.

    PubMed

    Esco, M A; Wang, Z; McDermott, M L; Kurpakus-Wheater, M

    2001-11-01

    Laminin 5 functions to promote cell-matrix adhesion and therefore is hypothesized to abrogate apoptosis initiated through the loss of epithelial cell contact with extracellular matrix. Laminin 5 levels are decreased in epithelial cells cultured in a hypoxic environment. Exposure of epithelial cells to hypoxia may induce apoptotic pathways transmitted through changes in mitochondrial membrane potential. Using an apoptosis assay based on mitochondrial membrane integrity, the effect of hypoxia (2% oxygen) on human corneal epithelial cell viability was determined. Both a virally transformed corneal epithelial cell line and third passage corneal epithelial cells were resistant to hypoxia-mediated apoptosis for up to 5 days in culture. However, at 7 days in culture, a statistically significant increase in apoptosis was noted in hypoxic corneal epithelial cells compared to normoxic (20% oxygen) controls. Increased apoptosis in hypoxic epithelium at 7 days in culture correlated with decreased deposition of laminin 5 into the extracellular matrix, as determined by western blot analysis and immunofluorescence microscopy. Additionally, the extracellular processing of the alpha3 and gamma2 chains of laminin 5 was negatively impacted by corneal epithelial cell exposure to hypoxia for 7 days. Treatment of human corneal epithelial cells cultured in 20% oxygen with function-inhibiting antibodies to laminin 5 for 2 or 3 days resulted in a statistically significant decrease in proliferation, and concomitant increase in apoptosis, compared with untreated normoxic controls. Based on these results, it appears that mechanisms of hypoxia-mediated apoptosis in human corneal epithelial cells may be initiated by the loss of processed laminin 5 in the extracellular matrix or by the loss of laminin 5-epithelial cell communication and transmitted through mitochondria.

  2. Human vomeronasal epithelium development: An immunohistochemical overview.

    PubMed

    Dénes, Lóránd; Pap, Zsuzsanna; Szántó, Annamária; Gergely, István; Pop, Tudor Sorin

    2015-06-01

    The vomeronasal organ (VNO) is the receptor structure of the vomeronasal system (VNS) in vertebrates. It is found bilaterally in the submucosa of the inferior part of the nasal septum. There are ongoing controversies regarding the functionality of this organ in humans. In this study we propose the immunohistochemical evaluation of changes in components of the human vomeronasal epithelium during foetal development. We used 45 foetuses of different age, which were included in three age groups. After VNO identification immunohistochemical reactions were performed using primary antibodies against the following: neuron specific enolase, calretinin, neurofilament, chromogranin, synaptophysin, cytokeratin 7, pan-cytokeratin and S100 protein. Digital slides were obtained and following colorimetric segmentation, surface area measurements were performed. The VNO was found in less than half of the studied specimens (42.2%). Neuron specific enolase and calretinin immunoexpression showed a decreasing trend with foetal age, while the other neural/neuroendocrine markers were negative in all specimens. Cytokeratin 7 expression increased with age, while Pan-Ctk had no significant variations. S100 protein immunoexpression also decreased around the VNO. The results of the present work uphold the theory of regression of the neuroepithelium that is present during initial stages of foetal development.

  3. Cellular Changes of the Corneal Epithelium and Stroma in Herpes Simplex Keratitis: An In Vivo Confocal Microscopy Study

    PubMed Central

    Hamrah, Pedram; Sahin, Afsun; Dastjerdi, Mohammad H.; Shahatit, Bashar M.; Bayhan, Hasan A.; Dana, Reza; Pavan-Langston, Deborah

    2012-01-01

    epithelial or stromal changes as compared to controls. Conclusions IVCM reveals profound HSK-induced changes in the superficial epithelium, as demonstrated by increase in cell size, decrease in cell density, and squamous metaplasia. We demonstrate that these changes strongly correlate with changes in corneal innervation. PMID:22608476

  4. Adrenergic regulation of cAMP/protein kinase A pathway in corneal epithelium and endothelium.

    PubMed

    Grueb, Matthias; Bartz-Schmidt, Karl Ulrich; Rohrbach, Jens Martin

    2008-10-01

    The G-protein-coupled receptor/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells. The aim of this study was to characterize subtypes of adrenergic G-protein-coupled receptors and their influence on cAMP concentration and PKA activity in bovine corneal epithelial and endothelial cells. Adrenergic receptors and PKA were studied using polyclonal antibodies. cAMP concentration was determined with an enzyme immunoassay, and PKA activity was estimated by the kinases consumption of adenosine triphosphate. In bovine corneal epithelial and endothelial cells, immunocytochemistry and Western blot were positive for alpha(1)-, alpha(2A)-, beta(1)- and beta(2)-adrenergic receptors. Stimulation of corneal epithelial and endothelial beta-adrenoceptors with isoprenaline led to a dose-dependent increase in cAMP concentration and activation of PKA. Stimulation of corneal alpha(2A)-adrenoceptors with brimonidine resulted in a dose-dependent decrease in cAMP concentration and the inhibition of PKA activity. In corneal epithelial and endothelial cells, beta-adrenergic stimulation leads to activation of PKA via stimulation of adenylyl cyclase, and alpha(2A)-adrenoceptor stimulation inhibits PKA activity via inhibition of adenylyl cyclase. Stimulation and inhibition of the corneal cAMP-PKA pathway may play a role in important corneal functions such as wound healing or homeostasis. Long-term therapy with alpha(2A)-agonists or beta-antagonists may influence these functions in a currently unknown way. Copyright 2008 S. Karger AG, Basel.

  5. Pseudomonas aeruginosa lipopolysaccharide binds galectin-3 and other human corneal epithelial proteins.

    PubMed Central

    Gupta, S K; Masinick, S; Garrett, M; Hazlett, L D

    1997-01-01

    The aim of this study was to test whether galectin-3 is present in human corneal epithelium and whether lipopolysaccharide (LPS) purified from Pseudomonas aeruginosa ATCC 19660 binds to this animal lectin and/or to another human corneal epithelial protein(s) (HCEP) and to confirm which component of LPS (inner or outer core or lipid A) is important in bacterial binding by using the eye in organ culture. LPS isolated and purified from P. aeruginosa ATCC 19660 and a commercial LPS (serotype 10) differed in polyacrylamide gel analysis but bound similarly to blotted HCEP. Binding was determined to be a receptor-ligand type of interaction by the solid-phase assay, because it was both specific and saturable. Several LPS binding proteins in HCEP were identified by an overlay method. Western blotting with antibody against galectin-3 revealed the presence of this protein in both freshly isolated and cultured transformed human corneal epithelium. Binding inhibition assays showed that antibody specific for the outer core region of LPS and an anti-galectin antibody significantly inhibited bacterial binding in vitro. These data provide further evidence that LPS is an important adhesin of P. aeruginosa, that it binds to protein receptor molecules in HCEP, that one of the LPS binding proteins is galectin-3, and that the outer core portion of the molecule appears to be critical for LPS binding to the eye. PMID:9199445

  6. Self-organized centripetal movement of corneal epithelium in the absence of external cues

    PubMed Central

    Lobo, Erwin P.; Delic, Naomi C.; Richardson, Alex; Raviraj, Vanisri; Halliday, Gary M.; Di Girolamo, Nick; Myerscough, Mary R.; Lyons, J. Guy

    2016-01-01

    Maintaining the structure of the cornea is essential for high-quality vision. In adult mammals, corneal epithelial cells emanate from stem cells in the limbus, driven by an unknown mechanism towards the centre of the cornea as cohesive clonal groups. Here we use complementary mathematical and biological models to show that corneal epithelial cells can self-organize into a cohesive, centripetal growth pattern in the absence of external physiological cues. Three conditions are required: a circumferential location of stem cells, a limited number of cell divisions and mobility in response to population pressure. We have used these complementary models to provide explanations for the increased rate of centripetal migration caused by wounding and the potential for stem cell leakage to account for stable transplants derived from central corneal tissue, despite the predominantly limbal location of stem cells. PMID:27499113

  7. Self-organized centripetal movement of corneal epithelium in the absence of external cues

    NASA Astrophysics Data System (ADS)

    Lobo, Erwin P.; Delic, Naomi C.; Richardson, Alex; Raviraj, Vanisri; Halliday, Gary M.; di Girolamo, Nick; Myerscough, Mary R.; Lyons, J. Guy

    2016-08-01

    Maintaining the structure of the cornea is essential for high-quality vision. In adult mammals, corneal epithelial cells emanate from stem cells in the limbus, driven by an unknown mechanism towards the centre of the cornea as cohesive clonal groups. Here we use complementary mathematical and biological models to show that corneal epithelial cells can self-organize into a cohesive, centripetal growth pattern in the absence of external physiological cues. Three conditions are required: a circumferential location of stem cells, a limited number of cell divisions and mobility in response to population pressure. We have used these complementary models to provide explanations for the increased rate of centripetal migration caused by wounding and the potential for stem cell leakage to account for stable transplants derived from central corneal tissue, despite the predominantly limbal location of stem cells.

  8. Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium.

    PubMed

    Uchino, Yuichi; Shimmura, Shigeto; Miyashita, Hideyuki; Taguchi, Tetsushi; Kobayashi, Hisatoshi; Shimazaki, Jun; Tanaka, Junzo; Tsubota, Kazuo

    2007-04-01

    Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis. (c) 2006 Wiley Periodicals, Inc.

  9. Inhibition of corneal neovascularization with the combination of bevacizumab and plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18 (p-PEDF-SAINT-18) vector in a rat corneal experimental angiogenesis model.

    PubMed

    Kuo, Chien-Neng; Chen, Chung-Yi; Chen, San-Ni; Yang, Lin-Cheng; Lai, Li-Ju; Lai, Chien-Hsiung; Chen, Miao-Fen; Hung, Chia-Hui; Chen, Ching-Hsein

    2013-04-16

    Bevacizumab, a 149-kDa protein, is a recombinant humanized monoclonal antibody to VEGF. PEDF, a 50-kDa glycoprotein, has demonstrated anti-vasopermeability properties. In this study, we demonstrated that the combination of bevacizumab and plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18(p-PEDF-SAINT-18) has a favorable antiangiogenic effect on corneal NV. Four groups(Group A: 0 μg + 0 μg, B: 0.1 μg + 0.1 μg, C: 1 μg + 1 μg, and D: 10 μg + 10 μg) of bevacizumab + p-PEDF-SAINT-18 were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus on the temporal side. Then, 1 μgof p-bFGF-SAINT-18 was prepared and implanted into the rat corneal stroma 1.5 mm from the limbus on the same side. The inhibition of NV was observed and quantified from days 1 to 60. Biomicroscopic examination, western blot analysis and immunohistochemistry were used to analyze the 18-kDa bFGF, 50-kDa PEDF and VEGF protein expression. No inhibition activity for normal limbal vessels was noted. Subconjunctival injection with the combination of bevacizumab and p-PEDF-SAINT-18 successfully inhibited corneal NV.The bFGF and PEDF genes were successfully expressed as shown by western blot analysis,and a mild immune response to HLA-DR was shown by immunohistochemistry. We concluded that the combination of bevacizumab and p-PEDF-SAINT-18 may have more potent and prolonged antiangiogenic effects, making it possible to reduce the frequency of subconjunctival bevacizumab administration combined with a relatively safe profile and low toxicity.

  10. Immunological activity to different corneal antigens in patients with corneal diseases.

    PubMed Central

    Brinkman, C J; Treffers, W F; Broekhuyse, R M

    1979-01-01

    Patients suffering from various corneal diseases and waiting for keratoplasty have been immunologically investigated in order to establish sensitisation to corneal antigens. The presence of lymphocytes sensitised to the soluble from human corneas, bovine corneal epithelium, and bovine corneal stroma, which all possess common antigenicity, could be demonstrated in 30%, 50%, and 23%, respectively, of all patients. In none of these patients could a positive plasma antibody titre to human corneal antigens be detected. The results suggest the dominance of T-lymphocyte activity. No correlation was found between the degree of corneal vascularisation and the presence of sensitised lymphocytes to human corneal antigens. Arrangement of the patients according to diagnosis showed that especially those suffering from herpes simplex virus keratitis reacted positively to human corneal antigens. A possible explanation is given. Lymphocytes of controls showed no or only very low stimulation with the soluble fractions of human corneas or bovine corneal stromas. The soluble fraction of bovine corneal epithelium stimulated the lymphocytes of 6 out of 19 controls. The elimination of the donor corneal epithelium before transplantation may be beneficial in view of the involvement of histocompatibility antigens. Images PMID:116673

  11. Wound-healing effect of micronized sacchachitin (mSC) nanogel on corneal epithelium

    PubMed Central

    Chen, Ray-Neng; Lee, Lin-Wen; Chen, Ling-Chun; Ho, Hsiu-O; Lui, Shiao-Chuan; Sheu, Ming-Thau; Su, Ching-Hua

    2012-01-01

    The extraction residue of the Ganoderma fruiting body, named sacchachitin, has been demonstrated to have the potential to enhance cutaneous wound healing by inducing cell proliferation. In this study, a nanogel formed from micronized sacchachitin (mSC) was investigated for the potential treatment of superficial chemical corneal burns. Reportedly, mSC has been produced successfully and its chemical properties confirmed, and physical and rheological properties characterized. An in vitro cell proliferation study has revealed that at the concentrations of 200, 300, and 400 μg/mL, mSC nanogel significantly increased Statens Seruminstitut rabbit corneal (SIRC) cell proliferation after 24 hours of incubation. In cell migration assay, migration of SIRC cell to wound closure was observed after 24 hours of incubation with the addition of 200 μg/mL mSC of nanogel. In an animal study, acceleration of corneal wound healing was probably due to the inhibition of proteolysis. In conclusion, the findings of this study substantiate the potential application of sacchachitin in the form of mSC nanogel for the treatment of superficial corneal injuries. PMID:22956870

  12. Effect of non-steroidal anti-inflammatory drugs (NSAID) on the rabbit corneal epithelium studied by scanning electron microscopy.

    PubMed

    Stroobants, A; Fabre, K; Maudgal, P C

    2000-01-01

    We investigated the effect of 6 commercially available non-steroidal anti-inflammatory drug (NSAID) eye drops on the normal corneal epithelium of rabbits. Each drug was instilled into both eyes of 2 rabbits, 5 times a day, for 5 consecutive days. Two additional corneas of one rabbit, without any treatment, served as control. After treatment, the corneas were excised and processed for scanning electron microscopic evaluation. The epithelial changes induced by the drugs were graded by an empirical score system. All test compounds caused alterations in the cell membranes and surface microvilli, or even exfoliation and necrosis of surface cells. The extent of cell damage appeared to be related to the active ingredient in the eye drops, the pH of the solution, and the constituents of the vehicle, especially the type of preservative used.

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

    PubMed

    Verstraelen, Jessica; Reichl, Stephan

    2014-07-07

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

  14. Morphological and biochemical evaluation for rigid gas permeable contact lens extended wear on rabbit corneal epithelium.

    PubMed

    Ichijima, H; Ohashi, J; Petroll, W M; Cavanagh, H D

    1993-04-01

    We studied the effects of 24-hour wear of rigid gas permeable (RGP) contact lenses of varying oxygen transmissibilities on the rabbit cornea by measuring concomitant lactate dehydrogenase (LDH) activity in tears and by in vivo tandem scanning confocal microscopy (TSCM). We used a PMMA lens and rigid gas permeable (RGP) lenses that had Dk/L values ranging from 7 to 64 x 10(-9) (cm/sec) (mL O2/mL mmHg) and a uniform 0.15 mm thickness. After 6- and 24-hour contact lens wear, rabbit tear LDH activity increased according to the decrease in the Dk of RGP lenses. Tear LDH activity after 24 hours of lens wear was higher than after 6 hours. The observed increase in tear LDH activity was correlated with in vivo corneal epithelial morphology by tandem scanning confocal microscopy. The observed severity of desquamation and swelling of corneal epithelial cells was dependent upon the Dk/Ltotal of contact lenses worn, which directly related to the contact lens induced corneal hypoxia. Based on the results of this study, we conclude that: 1) a nap or accidental overnight wear of contact lenses with less than 20 x 10(-9) Dk/Ltotal could cause severe corneal epithelial damage; 2) the ultra high Dk lens appeared to alter the ocular surface least; and 3) TSCM accompanied with tear LDH assay is an objective, non-invasive in vivo method to assess the effect of contact lens wear on the ocular surface over time at the cellular level.

  15. Translational issues for human corneal endothelial tissue engineering.

    PubMed

    Soh, Yu Qiang; Peh, Gary S L; Mehta, Jodhbir S

    2017-09-01

    Corneal endothelial disorders collectively represent a significant healthcare burden in most developed nations, and corneal transplantation is currently the only treatment available for patients with poor visual acuity and corneal blindness secondary to endothelial failure. Although vision in these patients can be restored by transplantation, the global demand for donor human corneas is far in excess of what can be provided for by eye banks around the world, and this deficit is set to increase with an ageing global population. As such, there has been a pressing need to explore novel and more sustainable options for the treatment of corneal endothelial diseases. In recent years, significant progress has been made not only in the development of corneal endothelial cell culture techniques, but also in the exploration of various translational strategies. Considered together, we are now much closer to attaining success in the treatment of corneal endothelial diseases via a cell-based, tissue-engineering approach. The aim of this review article is to provide an update of the translational issues currently facing human corneal endothelial cell therapy. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  16. Tear lipocalin: evidence for a scavenging function to remove lipids from the human corneal surface.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Prasher, Pawan; Yusifov, Taleh N; Glasgow, Ben J

    2005-10-01

    Lipid contamination of the cornea may create an unwettable surface and result in desiccation of the corneal epithelium. Tear lipocalin (TL), also known as lipocalin-1, is the principal lipid-binding protein in tears. TL has been shown to scavenge lipids from hydrophobic surfaces. The hypothesis that TL can remove contaminating fatty acids and phospholipids from the human corneal surface was tested. TL was purified from pooled human tear samples by size exclusion and ion exchange chromatographies. Tears depleted of TL were reconstituted from fractions eluted by size exclusion chromatography that did not contain TL. Fresh and formalin-fixed human corneas were obtained from exenteration specimens. Fluorescent analogs of either palmitic acid or phosphatidylcholine were applied to the corneal epithelial surface. Tears, TL, or tears depleted of TL were applied over the corneas, and spectrofluorometry and fluorescent stereomicroscopy were used to monitor the removal of fluorescent lipids. Tears used in the experiments were then fractionated by size exclusion chromatography to determine the component of tears associated with fluorescent lipids. Significant enhancement of fluorescence for 16AP and NBD C(6)-HPC was evident in solutions incubated with whole tears and purified TL but not with tears depleted of TL for fixed and unfixed corneas. After the experiment, size exclusion fractions of tears showed that the fluorescence component coeluted with TL. TL scavenges lipids from the human corneal surface and delivers them into the aqueous phase of tears. TL may have an important role in removing lipids from the corneal surface to maintain the wettability and integrity of the ocular surface.

  17. Multi-layered silk film co-culture system for human corneal epithelial and stromal stem cells.

    PubMed

    Gosselin, Emily A; Torregrosa, Tess; Ghezzi, Chiara E; Mendelsohn, Alexandra C; Gomes, Rachel; Funderburgh, James L; Kaplan, David L

    2017-06-10

    With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one co-culture system, in order to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these 3D tissue systems in vitro. Co-culture conditions were first optimized, including the medium, air/liquid interface culture, and surface topography and chemistry of biomaterial scaffold films based on silk protein. The silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs in this in vitro system were studied, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by RT-qPCR. The incorporation of both cell types into the co-culture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk films for corneal epithelial culture were optimized to combine a 4.0 micron-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in co-culture on the optimized silk scaffolds. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  18. Nonthermal Dielectric Barrier Discharge (DBD) Plasma Suppresses Herpes Simplex Virus Type 1 (HSV-1) Replication in Corneal Epithelium

    PubMed Central

    Alekseev, Oleg; Donovan, Kelly; Limonnik, Vladimir; Azizkhan-Clifford, Jane

    2014-01-01

    Purpose Herpes keratitis (HK) is the leading cause of cornea-derived and infection-associated blindness in the developed world. Despite the availability of effective antivirals, some patients develop refractory disease, drug-resistant infection, and topical toxicity. A nonpharmaceutical treatment modality may offer a unique advantage in the management of such cases. This study investigated the antiviral effect of nonthermal dielectric barrier discharge (DBD) plasma, a partially ionized gas that can be applied to organic substances to produce various biological effects. Methods Human corneal epithelial cells and explanted corneas were infected with herpes simplex virus type 1 (HSV-1) and exposed to culture medium treated with nonthermal DBD plasma. The extent of infection was measured by plaque assay, quantitative PCR, and Western blot. Corneal toxicity assessment was performed with fluorescein staining, histologic examination, and 8-OHdG detection. Results Application of DBD plasma–treated medium to human corneal epithelial cells and explanted corneas produced a dose-dependent reduction of the cytopathic effect, viral genome replication, and the overall production of infectious viral progeny. Toxicity studies showed lack of detrimental effects in explanted human corneas. Conclusions Nonthermal DBD plasma substantially suppresses corneal HSV-1 infection in vitro and ex vivo without causing pronounced toxicity. Translational Relevance Nonthermal plasma is a versatile tool that holds great biomedical potential for ophthalmology, where it is being investigated for wound healing and sterilization and is already in use for ocular microsurgery. The anti-HSV-1 activity of DBD plasma demonstrated here could be directly translated to the clinic for use against drug-resistant herpes keratitis. PMID:24757592

  19. Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation

    PubMed Central

    Wiechmann, Allan F.; Ceresa, Brian P.; Howard, Eric W.

    2014-01-01

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

  20. [Effect of beta-adrenoreceptor blockade on cell division processes in the corneal and tongue epithelium of white rats with chronic oxygen starvation].

    PubMed

    Vdovenko, S V; Timoshin, S S

    1985-01-01

    A study was made of the changes in the mitotic activity, DNA synthesis, and the number of pathological mitoses after administration of the beta-adrenoblocker propranolol in the corneal and tongue epithelium of white rats kept in pressure chamber for 7 days (4 h daily) at a "height" of 9000 meters. The mitotic and the label indices (inclusion of 3H-thymidine by the epithelium nuclei) were analysed for mitotic activity and DNA synthesis, respectively. The experiments showed that the mitotic activity, DNA synthesis, the number of pathological mitoses were stabilized due to the propranolol administration.

  1. Corneal repair by human corneal keratocyte-reprogrammed iPSCs and amphiphatic carboxymethyl-hexanoyl chitosan hydrogel.

    PubMed

    Chien, Yueh; Liao, Yi-Wen; Liu, Dean-Mo; Lin, Heng-Liang; Chen, Shih-Jen; Chen, Hen-Li; Peng, Chi-Hsien; Liang, Chang-Min; Mou, Chung-Yuan; Chiou, Shih-Hwa

    2012-11-01

    Induced pluripotent stem cells (iPSCs) have promising potential in regenerative medicine, but whether iPSCs can promote corneal reconstruction remains undetermined. In this study, we successfully reprogrammed human corneal keratocytes into iPSCs. To prevent feeder cell contamination, these iPSCs were cultured onto a serum- and feeder-free system in which they remained stable through 30 passages and showed ESC-like pluripotent property. To investigate the availability of iPSCs as bioengineered substitutes in corneal repair, we developed a thermo-gelling injectable amphiphatic carboxymethyl-hexanoyl chitosan (CHC) nanoscale hydrogel and found that such gel increased the viability and CD44+proportion of iPSCs, and maintained their stem-cell like gene expression, in the presence of culture media. Combined treatment of iPSC with CHC hydrogel (iPSC/CHC hydrogel) facilitated wound healing in surgical abrasion-injured corneas. In severe corneal damage induced by alkaline, iPSC/CHC hydrogel enhanced corneal reconstruction by downregulating oxidative stress and recruiting endogenous epithelial cells to restore corneal epithelial thickness. Therefore, we demonstrated that these human keratocyte-reprogrammed iPSCs, when combined with CHC hydrogel, can be used as a rapid delivery system to efficiently enhance corneal wound healing. In addition, iPSCs reprogrammed from corneal surgical residues may serve as an alternative cell source for personalized therapies for human corneal damage. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Modulating Endogenous Electric Currents in Human Corneal Wounds—A Novel Approach of Bioelectric Stimulation Without Electrodes

    PubMed Central

    Reid, Brian; Graue-Hernandez, Enrique O.; Mannis, Mark J.; Zhao, Min

    2011-01-01

    Purpose To measure electric current in human corneal wounds and test the feasibility of pharmacologically enhancing the current to promote corneal wound healing. Methods Using a noninvasive vibrating probe, corneal electric current was measured before and after wounding of the epithelium of donated postmortem human corneas. The effects of drug aminophylline and chloride-free solution on wound current were also tested. Results Unwounded cornea had small outward currents (0.07 μA/cm2). Wounding increased the current more than 5 fold (0.41 μA/cm2). Monitoring the wound current over time showed that it seemed to be actively regulated and maintained above normal unwounded levels for at least 6 hours. The time course was similar to that previously measured in rat cornea. Drug treatment or chloride-free solution more than doubled the size of wound currents. Conclusions Electric current at human corneal wounds can be significantly increased with aminophylline or chloride-free solution. Because corneal wound current directly correlates with wound healing rate, our results suggest a role for chloride-free and/or aminophylline eyedrops to enhance healing of damaged cornea in patients with reduced wound healing such as the elderly or diabetic patient. This novel approach offers bioelectric stimulation without electrodes and can be readily tested in patients. PMID:21099404

  3. Characterization of Side Population Cells from Human Airway Epithelium

    PubMed Central

    Hackett, Tillie-Louise; Shaheen, Furquan; Johnson, Andrew; Wadsworth, Samuel; Pechkovsky, Dmitri V.; Jacoby, David B.; Kicic, Anthony; Stick, Stephen M.; Knight, Darryl A.

    2010-01-01

    The airway epithelium is the first line of contact with the inhaled external environment and is continuously exposed to and injured by pollutants, allergens, and viruses. However, little is known about epithelial repair and in particular the identity and role of tissue resident stem/progenitor cells that may contribute to epithelial regeneration. The aims of the present study were to identify, isolate, and characterize side population (SP) cells in human tracheobronchial epithelium. Epithelial cells were obtained from seven nontransplantable healthy lungs and four asthmatic lungs by pronase digestion. SP cells were identified by verapamil-sensitive efflux of the DNA-binding dye Hoechst 33342. Using flow cytometry, CD45− SP, CD45+ SP, and non-SP cells were isolated and sorted. CD45− SP cells made up 0.12% ± 0.01% of the total epithelial cell population in normal airway but 4.1% ± 0.06% of the epithelium in asthmatic airways. All CD45− SP cells showed positive staining for epithelial-specific markers cytokeratin-5, E-cadherin, ZO-1, and p63. CD45− SP cells exhibited stable telomere length and increased colony-forming and proliferative potential, undergoing population expansion for at least 16 consecutive passages. In contrast with non-SP cells, fewer than 100 CD45− SP cells were able to generate a multilayered and differentiated epithelium in air-liquid interface culture. SP cells are present in human tracheobronchial epithelium, exhibit both short- and longterm proliferative potential, and are capable of generation of differentiated epithelium in vitro. The number of SP cells is significantly greater in asthmatic airways, providing evidence of dysregulated resident SP cells in the asthmatic epithelium. PMID:18653771

  4. Detection of human cytomegalovirus in normal and neoplastic breast epithelium

    PubMed Central

    2010-01-01

    Introduction Human cytomegalovirus (HCMV) establishes a persistent life-long infection, and can cause severe pathology in the fetus and the immunocompromised host[1]. Breast milk is the primary route of transmission in humans worldwide, and breast epithelium is thus a likely site of persistent infection and/or reactivation, though this phenomenon has not previously been demonstrated. Increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. We hypothesized that persistent HCMV infection occurs in normal adult breast epithelium and that persistent viral expression might be associated with normal and neoplastic ductal epithelium. Methods Surgical biopsy specimens of normal breast (n = 38) breast carcinoma (n = 39) and paired normal breast from breast cancer patients (n = 21) were obtained. Specimens were evaluated by immunohistochemistry, in situ hybridization, PCR and DNA sequencing for evidence of HCMV antigens and nucleic acids. Results We detected HCMV expression specifically in glandular epithelium in 17/27 (63%) of normal adult breast cases evaluated. In contrast, HCMV expression was evident in the neoplastic epithelium of 31/32 (97%) patients with ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) cases evaluated (p = 0.0009). Conclusions These findings are the first to demonstrate that persistent HCMV infection occurs in breast epithelium in a significant percentage of normal adult females. HCMV expression was also evident in neoplastic breast epithelium in a high percentage of normal and neoplastic breast tissues obtained from breast cancer patients, raising the possibility that viral infection may be involved in the neoplastic process. PMID:21429243

  5. Transcription factor TCF4 maintains the properties of human corneal epithelial stem cells.

    PubMed

    Lu, Rong; Qu, Yangluowa; Ge, Jian; Zhang, Lili; Su, Zhitao; Pflugfelder, Stephen C; Li, De-Quan

    2012-04-01

    TCF4, a key transcription factor of Wnt signaling system, has been recently found to be essential for maintaining stem cells. However, its signaling pathway is not well elucidated. This study was to explore the functional roles and signaling pathway of TCF4 in maintaining adult stem cell properties using human corneal epithelial stem cells as a model. With immunofluorescent staining and real-time polymerase chain reaction, we observed that TCF4 was exclusively expressed in the basal layer of human limbal epithelium where corneal epithelial stem cells reside. TCF4 was found to be well colocalized with ABCG2 and p63, two recognized epithelial stem/progenitor cell markers. Using in vitro culture models of primary human corneal epithelial cells, we revealed that TCF4 mRNA and protein were upregulated by cells in exponential growth stage, and RNA interference by small interfering RNA-TCF4 (10-50 nM) transfection blocked TCF4 signaling and suppressed cell proliferation as measured by WST-1 assay. TCF4 silence was found to be accompanied by downregulated proliferation-associated factors p63 and survivin, as well as upregulated cyclin-dependent kinase inhibitor 1C (p57). By creating a wound healing model in vitro, we identified upregulation and activation of β-catenin/TCF4 with their protein translocation from cytoplasm to nuclei, as evaluated by reverse transcription-quantitative real-time polymerase chain reaction, immunostaining, and Western blotting. Upregulated p63/survivin and downregulated p57 were further identified to be TCF4 downstream molecules that promote cell migration and proliferation in wound healing process. These findings demonstrate that transcription factor TCF4 plays an important role in determining or maintaining the phenotype and functional properties of human corneal epithelial stem cells. Copyright © 2012 AlphaMed Press.

  6. Safety and Efficacy of Epithelium-On Corneal Collagen Cross-Linking Using a Multifactorial Approach to Achieve Proper Stromal Riboflavin Saturation

    PubMed Central

    Stojanovic, Aleksandar; Chen, Xiangjun; Jin, Nan; Zhang, Ting; Stojanovic, Filip; Raeder, Sten; Utheim, Tor Paaske

    2012-01-01

    Purpose. To evaluate the efficacy and safety of epithelium-on corneal collagen cross-linking (CXL) using a multifactorial approach to achieve proper stromal riboflavin saturation. Methods. This non-randomized retrospective study comprised 61 eyes with progressive keratoconus treated with epithelium-on CXL. Chemical epithelial penetration enhancement (benzalkonium chloride-containing local medication and hypotonic riboflavin solution), mechanical disruption of the superficial epithelium, and prolongation of the riboflavin-induction time until verification of stromal saturation were used before the UVA irradiation. Uncorrected and corrected distance visual acuity (UDVA, CDVA), refraction, corneal topography, and aberrometry were evaluated at baseline and at 1, 3, 6, and 12 months postoperative. Results. At 12-month, UDVA and CDVA improved significantly. None of the eyes lost lines of CDVA, while 27.4% of the eyes gained 2 or more lines. Mean spherical equivalent decreased by 0.74 D, and mean cylindrical reduction was 1.15 D. Irregularity index and asymmetry from Scheimpflug-based topography and Max-K at the location of cone from Placido-based topography showed a significant decrease. Higher-order-aberration data demonstrated a slight reduction in odd-order aberrations S 3, 5,7 (P = 0.04). Postoperative pain without other complications was recorded. Conclusion. Epithelium-on CXL with our novel protocol appeared to be safe and effective in the treatment of progressive keratoconus. PMID:22900147

  7. Beta-adrenergic receptor agonists delay while antagonists accelerate epithelial wound healing: evidence of an endogenous adrenergic network within the corneal epithelium.

    PubMed

    Pullar, Christine E; Zhao, Min; Song, Bing; Pu, Jin; Reid, Brian; Ghoghawala, Shahed; McCaig, Colin; Isseroff, R Rivkah

    2007-04-01

    Wound healing is a complex and well-orchestrated biological process. Corneal epithelial cells (CECs) must respond quickly to trauma to rapidly restore barrier function and protect the eye from noxious agents. They express a high level of beta2-adrenergic receptors but their function is unknown. Here, we report the novel finding that they form part of a regulatory network in the corneal epithelium, capable of modulating corneal epithelial wound repair. Beta-adrenergic receptor agonists delay CEC migration via a protein phosphatase 2A-mediated mechanism and decrease both electric field-directed migration and corneal wound healing. Conversely, beta-adrenergic receptor antagonists accelerate CEC migration, enhance electric field-mediated directional migration, and promote corneal wound repair. We demonstrate that CECs express key enzymes required for epinephrine (beta-adrenergic receptor agonist) synthesis in the cytoplasm and can detect epinephrine in cell extracts. We propose that the mechanism for the pro-motogenic effect of the beta-adrenergic antagonist is blockade of the beta2-adrenergic receptor preventing autocrine catecholamine binding. Further investigation of this network will improve our understanding of one of the most frequently prescribed class of drugs. (c) 2007 Wiley-Liss, Inc.

  8. Methods for culture of human corneal and conjunctival epithelia.

    PubMed

    Spurr-Michaud, Sandra J; Gipson, Ilene K

    2013-01-01

    The surface of the eye is exposed to the outside world and is, thus, subject to surface abrasion, infections, and drying, cicatrizing diseases. Availability of in vitro methods for culture of the human corneal and conjunctival epithelia, which cover the ocular surface, is therefore important in understanding the biology of these epithelia and their response to disease/infections, as well as for providing human-relevant models for preclinical testing of potential therapeutic agents. The ensuing chapter describes several methods for primary culture of both corneal and conjunctival epithelia and culture of immortalized cell lines, and methods employed to induce differentiation in the cultured epithelia.

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

    PubMed

    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.

  10. Effects of formaldehyde on normal xenotransplanted human tracheobronchial epithelium.

    PubMed Central

    Ura, H.; Nowak, P.; Litwin, S.; Watts, P.; Bonfil, R. D.; Klein-Szanto, A. J.

    1989-01-01

    Epithelial cells obtained from autopsies of full-term fetuses or infants less than 1 year old were isolated, amplified in primary cultures and inoculated in deepithelialized rat tracheas. These tracheas were then sealed and transplanted subcutaneously into irradiated athymic nude mice. Four weeks after transplantation the tracheal lumen was completely covered by epithelium, most of which was of mucociliary respiratory type. At this stage, tracheal transplants containing tracheobronchial epithelium from 20 different donors were exposed to silastic devices containing 0, 0.5, 1 and 2 mg paraformaldehyde. The tracheal transplants were examined histologically at 2, 4, 8, and 16 weeks after transplantation. Before sacrifice, all animals were injected with a single pulse of tritiated thymidine. Important epithelial alterations could be seen in the formaldehyde treated transplants with a maximum effect visible at 2 weeks after exposure. The highest dose of 2 mg produced, in most cases, numerous areas of epithelial erosion and inflammation whereas this effect was not as evident with the lower doses. All doses produced areas of hyperplastic epithelium alternating with areas of pleomorphic-atrophic epithelium. Although the differences in predominance of different types of epithelium was not clearly dose-dependent, the labeling index (LI) showed dose dependence between 2 and 4 weeks after initiation of exposure. The maximum mean LI was three to four times higher than normal, although in some focal hyperplastic-metaplastic lesions the LI was increased up to 20 times. These studies show that formaldehyde, although toxic at higher doses, is able to elicit at lower doses a proliferative response of the human respiratory epithelium that is not preceded by a massive toxic effect. This response is similar, although less intense than that of the rat respiratory epithelium in which formaldehyde proved to be a carcinogen. Images Figure 2 Figure 5 PMID:2913828

  11. Safety and efficacy of epithelium removal and transepithelial corneal collagen crosslinking for keratoconus

    PubMed Central

    Shalchi, Z; Wang, X; Nanavaty, M A

    2015-01-01

    This review aims to assess the efficacy and safety of epithelial removal (ER) and transepithelial (TE) corneal collagen crosslinking (CXL) for the treatment of keratoconus. We used MEDLINE to identify all ER and TE CXL studies on keratoconic eyes (n≥20, follow-up ≥12 months). Ex vivo and studies for non-keratoconus indications or in conjunction with other procedures were excluded. Data on uncorrected (UDVA) and corrected (CDVA) distance visual acuity, refractive cylinder, maximum keratometry (Kmax), and adverse events were collected at the latest follow-up and 1 year. Only one randomised controlled trial (RCT) qualified inclusion. Forty-four ER and five TE studies were included. For logMAR UDVA, CDVA, mean spherical equivalent, refractive cylinder and Kmax, at latest follow-up 81, 85, 93, 62, and 93% ER studies vs 66.7, 80, 75, 33, and 40% TE studies reported improvement, respectively. Whereas at 1 year, 90, 59, and 91% ER studies vs 80, 50, and 25% TE studies reported improvement, respectively. The majority of studies showed reduced pachymetry in both groups. Treatment failure, retreatment rates, and conversion to transplantation were reported to be up to 33, 8.6, and 6.25%, respectively, in ER studies only. Stromal oedema, haze, keratitis, and scarring were only reported in ER studies, whereas endothelial cell counts remained variable in both groups. Both ER and TE studies showed improvement in visual acuity, refractive cylinder but Kmax worsened in most TE studies. Adverse events were reported more with ER studies. This review calls for more high quality ER and TE studies with comparable parameters for further assessment of safety and efficacy. PMID:25277300

  12. The effects of coronavirus on human nasal ciliated respiratory epithelium.

    PubMed

    Chilvers, M A; McKean, M; Rutman, A; Myint, B S; Silverman, M; O'Callaghan, C

    2001-12-01

    Human coronavirus (HCoV) accounts for 15-30% of common colds, but only one case report has described the effect of a coronavirus infection, that was asymptomatic, on human respiratory epithelium. The authors examined the effects of infection with HCoV on ciliary structure and function in healthy volunteers infected by intranasal inoculation with HCoV 229E. A further four volunteers were sham infected with ultraviolet-inactivated virus. Immediately before inoculation (day 0) and 3 days later (day 3), ciliated epithelium was obtained by brushing the inferior nasal turbinate. Ciliary beat frequency was determined and beat pattern analysed for evidence of dyskinesia (0=normal, 3=severely dyskinetic) using digital high-speed video photography. Ciliary ultrastructure was examined by transmission electron microscopy. Symptom diaries were kept for the duration of the study. All subjects inoculated with HCoV, including the three who did not develop symptoms of an upper respiratory tract infection, had disruption of their respiratory epithelium on day 3. Although there was no difference in the mean ciliary beat frequency between day 0 (11.3 Hz (95% confidence interval (CI): 8.6-14.0) and day 3 (9.4 Hz (95% CI 7.2-11.6)), there was a significant increase (p<0.05) in the ciliary dyskinesia score between day 0 (0.2 (95% CI 0-0.5)) and day 3 (1.1 (95% CI 0.5-1.7). In sham-infected subjects, no differences in epithelial integrity, or ciliary structure and function were found between day 0 and day 3. Inoculation of healthy volunteers with human coronavirus caused disruption of the ciliated epithelium and ciliary dyskinesia. This is likely to impair mucociliary clearance. Damage to the respiratory epithelium, due to human coronavirus infection, may occur without overt clinical symptoms.

  13. Elevated IGFBP3 levels in diabetic tears: a negative regulator of IGF-1 signaling in the corneal epithelium

    PubMed Central

    Wu, Yu-Chieh; Buckner, Benjamin R.; Zhu, Meifang; Cavanagh, H. Dwight; Robertson, Danielle M.

    2012-01-01

    Purpose To determine the ratio of IGFBP3:IGF-1 in normal and diabetic human tears, and in telomerase-immortalized human corneal epithelial cells (hTCEpi) cultured under elevated glucose conditions and to correlate these changes with total and phosphorylated levels of IGF-1R. Methods Tear samples were collected noninvasively from diabetic subjects and non-diabetic controls; corneal sensitivity was assessed using a Cochet-Bonnet Aesthesiometer. Conditioned media were collected following culture of hTCEpi cells in normal (5 mM) and elevated (25 mM) glucose conditions; mannitol was used as an osmotic control. IGFBP3, IGF-1, and phosphorylated IGF-1R levels were assessed by ELISA. IGFBP3 and IGF-1R mRNA were assessed by real time polymerase chain reaction (PCR). Total and phosphorylated IGF-1R expression in whole cell lysates was assessed by western blot. Results There was a 2.8-fold increase in IGFBP3 in diabetic tears compared to non-diabetic controls (P=0.006); IGF-1 levels were not significantly altered. No difference in corneal sensitivity was detected between groups. The concentration of IGFBP3 in tears was independent of IGF-1. Consistent with human tear measurements in vivo, IGFBP3 secretion was increased 2.2 fold (P<0.001) following culture of hTCEpi cells under elevated glucose conditions in vitro. Treatment with glucose and the mannitol control reduced IGFBP3 mRNA (P<0.001). Total IGF-1R levels were unchanged. The increase in the IGFBP3:IGF-1 ratio detected in diabetic tears compared to normal controls blocked phosphorylation of the IGF-1R by IGF-1 (P<0.001) when tested in vitro. Conclusions Taken together, these in vivo and confirmatory in vitro findings suggest that the observed increase in IGFBP3 found in human tears may attenuate IGF-1R signaling in the diabetic cornea. A long-term increase in IGFBP3 may contribute to epithelial compromise and the pathogenesis of ocular surface complications reported in diabetes. PMID:22482470

  14. Elevated IGFBP3 levels in diabetic tears: a negative regulator of IGF-1 signaling in the corneal epithelium.

    PubMed

    Wu, Yu-Chieh; Buckner, Benjamin R; Zhu, Meifang; Cavanagh, H Dwight; Robertson, Danielle M

    2012-04-01

    To determine the ratio of IGFBP3:IGF-1 in normal and diabetic human tears, and in telomerase-immortalized human corneal epithelial cells (hTCEpi) cultured under elevated glucose conditions and to correlate these changes with total and phosphorylated levels of IGF-1R. Tear samples were collected noninvasively from diabetic subjects and non-diabetic controls; corneal sensitivity was assessed using a Cochet-Bonnet Aesthesiometer. Conditioned media were collected following culture of hTCEpi cells in normal (5 mM) and elevated (25 mM) glucose conditions; mannitol was used as an osmotic control. IGFBP3, IGF-1, and phosphorylated IGF-1R levels were assessed by ELISA. IGFBP3 and IGF-1R mRNA were assessed by real-time polymerase chain reaction (PCR). Total and phosphorylated IGF-1R expression in whole cell lysates was assessed by western blot. There was a 2.8-fold increase in IGFBP3 in diabetic tears compared to non-diabetic controls (P=0.006); IGF-1 levels were not significantly altered. No difference in corneal sensitivity was detected between groups. The concentration of IGFBP3 in tears was independent of IGF-1. Consistent with human tear measurements in vivo, IGFBP3 secretion was increased 2.2 fold (P<0.001) following culture of hTCEpi cells under elevated glucose conditions in vitro. Treatment with glucose and the mannitol control reduced IGFBP3 mRNA (P<0.001). Total IGF-1R levels were unchanged. The increase in the IGFBP3:IGF-1 ratio detected in diabetic tears compared to normal controls blocked phosphorylation of the IGF-1R by IGF-1 (P<0.001) when tested in vitro. Taken together, these in vivo and confirmatory in vitro findings suggest that the observed increase in IGFBP3 found in human tears may attenuate IGF-1R signaling in the diabetic cornea. A long-term increase in IGFBP3 may contribute to epithelial compromise and the pathogenesis of ocular surface complications reported in diabetes. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. [Cytogenetic damage to the corneal epithelium of mice due to the in vivo exposure to ionizing radiation with different levels of linear energy transfer].

    PubMed

    Vorozhtsova, S V; Bulynina, T M; Molokanov, A G; Ivanov, A A

    2015-01-01

    Damages to corneal epithelium cells were studied in mice irradiated by protons with the energies of 10, 25, 50 and 645 MeV, 60Co γ-quanta and accelerated ions of boron, carbon and neon with the energies of 7.5; 2.5 and 6.0 MeV/nucleon, respectively. X-rays (180 keV) were used as a standard radiation. Animals were exposed to a single dose in the range from 25 to 760 cGy. The mitotic index and aberrant mitoses were counted in corneal preparations in 24 hrs after irradiation. No matter the type of radiation, the mitotic index had an inverse dose dependence, i.e. the higher the dose, the lower the mitotic index. Exposure to all types of radiation resulted in a sharp increase in the number of chromosomal aberrations in the corneal epithelium; frequency of aberrations was a function of dose and type of radiation. The number of chromosomal aberrations displayed a peculiar direct dose dependence irrespective of type of radiation; however, heavy ions of carbon and boron are the most damaging to the cytogenetic apparatus of epithelial cells. Protons at the Bragg peak and ensuing fall, and of 50 MeV also contribute to the production of chromosomal aberrations as compared with sparsely ionizing gamma- and X-rays and high-energy protons with low linear energy transfer. Coefficients of relative biological effectiveness were calculated based on the mitotic index and evidence of aberrant mitosis.

  16. Oxidative stress gradient in a medium during human corneal organ culture

    PubMed Central

    Johnsen-Soriano, Siv; Haug, Kristiane; Arnal, Emma; Peris-Martinez, Cristina; Moe, Morten C.

    2012-01-01

    Purpose Lipid peroxidation content was measured in an organ culture medium after one-week storage of human donor corneas. Moreover, the effects of the medium on oxidative stress, antioxidant capacity, and the proliferation of cultured human corneal cells were studied. Methods The medium was sampled from the upper and lower halves of storage vials and from controls (n=42). Malondialdehyde (MDA) was measured by high pressure liquid chromatography (HPLC). Cultured human corneal epithelium (CRL-11515) was exposed to different medium samples and monitored for changes in MDA (enzyme-linked immunosorbent assay [ELISA]), total antioxidant capacity (antioxidant assay kit), and proliferation (Ki-67). Results A significant increase in MDA was observed in the organ culture medium in the lower level of storage vials. The addition of this fraction to cultured cells increased MDA significantly after 3 days, and the medium from both levels significantly increased MDA after 7 days. The medium from both levels significantly decreased the total antioxidant capacity of the cells but did not affect proliferative activity. Conclusions An oxidative gradient with an evident biologic effect is established in the medium in vials during organ culture of human donor corneas. Donor tissue stored at the bottom or in lower levels of such vials is exposed to a significant amount of oxidative stress. PMID:22736949

  17. Microbes on the human vaginal epithelium

    PubMed Central

    Hyman, Richard W.; Fukushima, Marilyn; Diamond, Lisa; Kumm, Jochen; Giudice, Linda C.; Davis, Ronald W.

    2005-01-01

    Using solely a gene-based procedure, PCR amplification of the 16S ribosomal RNA gene coupled with very deep sequencing of the amplified products, the microbes on 20 human vaginal epithelia of healthy women have been identified and quantitated. The Lactobacillus content on these 20 healthy vaginal epithelia was highly variable, ranging from 0% to 100%. For four subjects, Lactobacillus was (virtually) the only bacterium detected. However, that Lactobacillus was far from clonal and was a mixture of species and strains. Eight subjects presented complex mixtures of Lactobacillus and other microbes. The remaining eight subjects had no Lactobacillus. Instead, Bifidobacterium, Gardnerella, Prevotella, Pseudomonas, or Streptococcus predominated. PMID:15911771

  18. Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts

    PubMed Central

    Yam, Gary Hin-Fai; Williams, Geraint P.; Setiawan, Melina; Yusoff, Nur Zahirah Binte M.; Lee, Xiao-wen; Htoon, Hla Myint; Zhou, Lei; Fuest, Matthias; Mehta, Jodhbir S.

    2017-01-01

    Laser refractive surgeries reshape corneal stroma to correct refractive errors, but unavoidably affect corneal nerves. Slow nerve regeneration and atypical neurite morphology cause desensitization and neuro-epitheliopathy. Following injury, surviving corneal stromal keratocytes (CSKs) are activated to stromal fibroblasts (SFs). How these two different cell types influence nerve regeneration is elusive. Our study evaluated the neuro-regulatory effects of human SFs versus CSKs derived from the same corneal stroma using an in vitro chick dorsal root ganglion model. The neurite growth was assessed by a validated concentric circle intersection count method. Serum-free conditioned media (CM) from SFs promoted neurite growth dose-dependently, compared to that from CSKs. We detected neurotrophic and pro-inflammatory factors (interleukin-8, interleukin-15, monocyte chemoattractant protein-1, eotaxin, RANTES) in SFCM by Bio-Plex Human Cytokine assay. More than 130 proteins in SFCM and 49 in CSKCM were identified by nanoLC-MS/MS. Proteins uniquely present in SFCM had reported neuro-regulatory activities and were predicted to regulate neurogenesis, focal adhesion and wound healing. Conclusively, this was the first study showing a physiological relationship between nerve growth and the metabolically active SFs versus quiescent CSKs from the same cornea source. The dose-dependent effect on neurite growth indicated that nerve regeneration could be influenced by SF density. PMID:28349952

  19. Novel Identity and Functional Markers for Human Corneal Endothelial Cells

    PubMed Central

    Bartakova, Alena; Alvarez-Delfin, Karen; Weisman, Alejandra D.; Salero, Enrique; Raffa, Gabriella A.; Merkhofer, Richard M.; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

    2016-01-01

    Purpose Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy. Methods Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays. Results Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs. Conclusions In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction. PMID:27196322

  20. Mitochondrial DNA common deletion in the human eye: a relation with corneal aging.

    PubMed

    Gendron, Sébastien P; Mallet, Justin D; Bastien, Nathalie; Rochette, Patrick J

    2012-01-01

    The most frequent mitochondrial DNA (mtDNA) mutation is a 4977 bp deletion known as the common deletion (mtDNA(CD4977)). mtDNA(CD4977) is related to skin photo-aging and to chronological aging of cells with high-energy demands such as neurons and muscle cells. The human eye contains both sun-exposed (cornea, iris) and high-energy demand structures (retina). In this study, we employed a highly sensitive quantitative PCR technique to determine mtDNA(CD4977) occurrence in different structures of the human eye. We found that the cornea, the most anterior structure of the eye, contains the highest amount of mtDNA(CD4977) (2.6%, 0.25% and 0.06% for the cornea, iris and retina, respectively). Within the cornea, mtDNA(CD4977) is almost exclusively found in the stroma, the cellular layer conferring transparency and rigidity to the human cornea (8.59%, 0.13% and 0.05% in the stroma, endothelium and epithelium, respectively). Moreover, we show that mtDNA(CD4977) accumulates with age in the corneal stroma. Taken together, our results suggest that mtDNA(CD4977) is related to photo-aging rather than chronological aging in the human eye. Similar to the involvement of mtDNA(CD4977) in skin photo-aging phenotypes, we believe that the clinical manifestations of corneal aging, including clouding and stiffening, are associated with the accumulation of mtDNA(CD4977) in the corneal stroma. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  1. Can Riboflavin Penetrate Stroma Without Disrupting Integrity of Corneal Epithelium in Rabbits? Iontophoresis and Ultraperformance Liquid Chromatography With Electrospray Ionization Tandem Mass Spectrometry.

    PubMed

    Novruzlu, Şahin; Türkcü, Ümmühani Özel; Kvrak, İbrahim; Kvrak, Şeyda; Yüksel, Erdem; Deniz, Nuriye Gökçen; Bilgihan, Ayşe; Bilgihan, Kamil

    2015-08-01

    To examine riboflavin concentrations in corneas and aqueous humor from rabbits with standard and transepithelial methods and iontophoresis without disrupting the integrity of the corneal epithelium before corneal collagen cross-linking. Twenty-four eyes of 12 adult New Zealand rabbits were used. They were assigned to 4 groups, each including 6 eyes. Group 1 was exposed to the standard method and given riboflavin 0.1% after epithelial debridement. Group 2 was exposed to the transepithelial method and given benzalkonium chloride (BAC), ethylenediaminetetraacetic acid (EDTA), trometamol (TRIS), hydroxypropylmethylcellulose (HPMC), and riboflavin 0.2% 3 times at 1.5-minute intervals followed by riboflavin 0.2%. Group 3 was given riboflavin 0.1% by using 1-mA electric current for 10 minutes with the help of iontophoresis without using substances disrupting the integrity of the corneal epithelium. Group 4 received the same treatment as did group 3, except that it was given riboflavin 0.2%. Following these treatments, riboflavin concentrations in aqueous humor and corneas were measured with ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). Riboflavin concentrations in the cornea and aqueous humor were higher in group 1 (42.4 ± 5.4 μg/g) than in the other groups. They were significantly higher in group 4 (34.2 ± 6.6 μg/g) than in group 2 (24.4 ± 1.2 μg/g) (P = 0.009) and group 3 (23.6 ± 6.1 μg/g) (P = 0.026). There was not a significant difference in corneal riboflavin concentrations between group 2 and group 3 (P = 0.937). Intrastromal and aqueous riboflavin concentrations after administration of riboflavin 0.2% through iontophoresis without disrupting the integrity of the corneal epithelium were lower than those after the standard method, but higher than those after the transepithelial method. In this study, in which riboflavin concentrations were measured with a very sensitive method

  2. Human Cornea Proteome: Identification and Quantitation of the Proteins of the Three Main Layers Including Epithelium, Stroma, and Endothelium

    PubMed Central

    2012-01-01

    Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC–MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses. PMID:22698189

  3. Scattering properties and transparency characterization of human corneal grafts

    NASA Astrophysics Data System (ADS)

    Casadessus, Olivier; Georges, Ga"lle; Siozade-Lamoine, Laure; Deumié, Carole; Conrath, John; Hoffart, Louis

    2011-06-01

    The cornea is the single human tissue being transparent. This unique property may be explained by the particular structure of the cornea, but the precise role of each of its constituents remains unsolved. On other matter, prior to corneal transplant, graft must be evaluated during a sorting procedure where a technician assesses of its transparency quality. Nevertheless, this criterion remains subjective and qualitative. This study proposes to combine 3D imagery using Full-Field Optical Coherence Tomography jointly with angular resolved scattering measurement to achieve a quantitative transparency characterization of the cornea. The OCT provides micrometric resolution structural information about the cornea, and we observe the evolution occurring when oedema develops within the tissue. Scattering properties are evaluated and compared parallely, as the transparency of the graft. A close link between the scattering intensity level of the cornea and its thickness is highlighted through this study. Furthermore, the three-dimensional imagery offers a view over the structural modifications leading to a change in transparency, and the combination with scattering properties measurement provides clues over the characteristic scale of scatterers to consider for a better understanding of corneal transparency evolution. Achieving an objective and quantified parameter for the transparency would be helpful for a more efficient corneal graft sorting, and may be able to detect the presence of localized wounds as the ones related to a previous refractive surgery. However, the study of graft nearly eligible for corneal transplant would be needed to confirm the results this study presents.

  4. Enhancing effects of sericin on corneal wound healing in Otsuka Long-Evans Tokushima fatty rats as a model of human type 2 diabetes.

    PubMed

    Nagai, Noriaki; Murao, Takatoshi; Ito, Yoshimasa; Okamoto, Norio; Sasaki, Masahiro

    2009-09-01

    The protein sericin is the main constituent of silk. We investigated the effects of sericin on corneal wound healing in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model for human type 2 diabetes. Corneal wounds were prepared by removal of the corneal epithelium, and documented using a TRC-50X equipped with a digital camera. Sericin solutions were instilled into the eyes of rats five times a day following corneal abrasion. Plasma glucose and triglycerides were determined using an Accutrend GCT. Cholesterol and insulin were measured using a Cholesterol E-Test Kit and ELISA Insulin Kit, respectively. The plasma levels of glucose, triglycerides, cholesterol and insulin in 38-week-old OLETF rats were significantly higher than in Long-Evans Tokushima Otsuka (LETO) rats used as normal controls, and the rate of corneal wound healing in OLETF rats was slower than in LETO rats. The corneal wounds of rats instilled with saline showed almost complete healing by 72 h after corneal epithelial abrasion. On the other hand, the corneal healing rate of OLETF rats instilled with 10% sericin solution was significantly higher than that of LETO rats instilled with saline, and the wounds showed almost complete healing at 48 h after abrasion. The corneal healing rate increased with increasing sericin concentration. The present study demonstrates that the corneal wound healing rate in OLETF rat is slower than in LETO rats, and the instillation of sericin solution has a potent effect in promoting wound healing and wound-size reduction in LETO and OLETF rats.

  5. Morphology of the epithelium of the lower rectum and the anal canal in the adult human.

    PubMed

    Tanaka, Eiichi; Noguchi, Tsuyoshi; Nagai, Kaoruko; Akashi, Yuichi; Kawahara, Katsunobu; Shimada, Tatsuo

    2012-06-01

    The anal canal is an important body part clinically. However, there is no agreement about the epithelium of the anal canal, the anal transitional zone (ATZ) epithelium in particular. The aim of this study is to clarify the structure of the epithelium of the human lower rectum and anal canal. Intact rectum and anus obtained from patients who underwent surgery for rectal carcinoma were examined by light and scanning electron microscopy (LM and SEM). By LM, three types of epithelium were observed in the anal canal: simple columnar epithelium, stratified squamous epithelium, and stratified columnar epithelium. The lower rectum was composed of simple columnar epithelium. SEM findings showed stratified squamous epithelium that consisted of squamous cells with microridges, changing to simple columnar epithelium consisting of columnar cells with short microvilli at the anorectal line. LM and SEM observations in a one-to-one ratio revealed that the area of stratified columnar epithelium based on LM corresponded to the anal crypt and sinus. In conclusion, the epithelium of the human anal canal was fundamentally composed of simple columnar epithelium and stratified squamous epithelium. We found no evidence of the ATZ.

  6. Human Corneal Endothelial Cells Expanded In Vitro Are a Powerful Resource for Tissue Engineering.

    PubMed

    Liu, Yongsong; Sun, Hong; Hu, Min; Zhu, Min; Tighe, Sean; Chen, Shuangling; Zhang, Yuan; Su, Chenwei; Cai, Subo; Guo, Ping

    2017-01-01

    Human corneal endothelial cells have two major functions: barrier function mediated by proteins such as ZO-1 and pump function mediated by Na-K-ATPase which help to maintain visual function. However, human corneal endothelial cells are notorious for their limited proliferative capability in vivo and are therefore prone to corneal endothelial dysfunction that eventually may lead to blindness. At present, the only method to cure corneal endothelial dysfunction is by transplantation of a cadaver donor cornea with normal corneal endothelial cells. Due to the global shortage of donor corneas, it is vital to engineer corneal tissue in vitro that could potentially be transplanted clinically. In this review, we summarize the advances in understanding the behavior of human corneal endothelial cells, their current engineering strategy in vitro and their potential applications.

  7. Human Corneal Endothelial Cells Expanded In Vitro Are a Powerful Resource for Tissue Engineering

    PubMed Central

    Liu, Yongsong; Sun, Hong; Hu, Min; Zhu, Min; Tighe, Sean; Chen, Shuangling; Zhang, Yuan; Su, Chenwei; Cai, Subo; Guo, Ping

    2017-01-01

    Human corneal endothelial cells have two major functions: barrier function mediated by proteins such as ZO-1 and pump function mediated by Na-K-ATPase which help to maintain visual function. However, human corneal endothelial cells are notorious for their limited proliferative capability in vivo and are therefore prone to corneal endothelial dysfunction that eventually may lead to blindness. At present, the only method to cure corneal endothelial dysfunction is by transplantation of a cadaver donor cornea with normal corneal endothelial cells. Due to the global shortage of donor corneas, it is vital to engineer corneal tissue in vitro that could potentially be transplanted clinically. In this review, we summarize the advances in understanding the behavior of human corneal endothelial cells, their current engineering strategy in vitro and their potential applications. PMID:28260988

  8. Immortalization of human corneal epithelial cells using simian virus 40 large T antigen and cell characterization.

    PubMed

    Kim, Cho-Won; Go, Ryeo-Eun; Lee, Geum-A; Kim, Chang Deok; Chun, Young-Jin; Choi, Kyung-Chul

    2016-01-01

    Primary cultures of human corneal epithelial (HCE) cells usually cease to grow after four or five passages. This result in a small cell yield for experiments such as the eye irritancy test represents a serious problem for human and animal corneal epithelial research. In the present study, we established an HCE cell line immortalized by simian virus 40 (SV40), a polyomavirus, and characterized the inherent morphologic and cytologic cell properties. Primary cultured HCE cells were infected with a SV40 large T antigen (SV40 T)-expressing retrovirus, and were selected using G418 solution, an aminoglycoside antibiotic. To ensure that the immortalized cell lines express SV40 T and cytokeratin-3, a corneal epithelial-specific marker, we conducted reverse-transcription (RT)-PCR and Western blot analysis. These cell lines continued to grow for more than 50 generations, exhibiting a cobble stone-like appearance similar to normal HCE cells and an increased proliferation rate compared to primary cultured HCE cells. RT-PCR results showed that the immortalized cell lines expressed SV40 T while the primary cultured cells did not. In the Western blot assay, protein levels of phosphorylated (Ser15) p53 protein were significantly decreased in the immortalized cell lines while the expression of total p53 protein was constant. In addition, expression of p21(cip1), a cell cycle protein, was down-regulated in the immortalized cells. Moreover, a cornea epithelium-specific marker, cytokeratin-3 (CK-3), was expressed at equal levels in the immortalized cells and primary HCE cells. Taken together, these results indicate that immortalized HCE cell lines were successfully established using the SV40-retroviral vector. These cells may be an excellent model for detecting the adverse effects of standard toxic materials and could replace the traditional eye irritation test as an animal-free alternative method. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Toxicity of natural tear substitutes in a fully defined culture model of human corneal epithelial cells.

    PubMed

    Geerling, G; Daniels, J T; Dart, J K; Cree, I A; Khaw, P T

    2001-04-01

    Serum and saliva have recently been advocated as natural tear substitutes for intractable aqueous-deficient dry eyes, but the effects of these fluids on corneal epithelium have not been well characterized. A laboratory study was performed in a defined test model to compare the toxicity of natural and pharmaceutical tear substitutes and to identify potentially toxic factors in natural tear substitutes, such as amylase, hypotonicity, and variations in preparation. Primary human corneal epithelial cells were cultured with defined keratinocyte serum-free medium. The cells were incubated with hypromellose (hydroxypropylmethylcellulose 0.3%) with and without benzalkonium chloride 0.01%, saliva with differing osmolalities, 100% serum, and 50% serum (1:1 vol/vol with chloramphenicol 0.5%) for varying times and concentrations. Toxicity was examined in four ways. Microvillous density was assessed with scanning electron microscopy. Cell membrane permeability and intracellular esterase activity were analyzed after staining with fluorescent calcein-AM/ethidium homodimer and cellular adenosine triphosphate (ATP) was quantified using a luciferin-luciferase-based assay. The toxicity ranking of the tear substitutes correlated in all assays. The ATP assay was the most sensitive, followed by ethidium cell permeability, and finally the esterase activity. Preserved hypromellose was more toxic than the unpreserved preparation. Among natural tear substitutes, natural saliva was most toxic. Isotonic saliva and 50% serum were of similar toxicity, and 100% serum was least toxic. Natural tear substitutes were-except for natural saliva-less toxic than unpreserved hypromellose. Hypotonicity, but not amylase, was the major toxic effect associated with saliva. The dilution of serum with chloramphenicol induced toxicity. This is the first toxicity study using human primary corneal epithelial cells cultured under fully defined conditions as an in vitro model. Cellular ATP is a sensitive parameter

  10. [CYSTEAMINE-INDUCED MODIFICATION OF CYTOGENETIC DAMAGES TO THE CORNEAL EPITHELIUM OF MICE EXPOSED TO CORPUSCULAR RADIATION WITH VARYING LINEAR TRANSFER ENERGIES].

    PubMed

    Vorozhtsova, S V; Bulynina, T M; Molokanov, A G; Ivanov, A A

    2015-01-01

    Cytogenetic damages to cells of the corneal epithelium were studied in mice exposed to protons (10, 25, 50 and 645 MeV), ions of boron, carbon and neon, and X-rays (180 keV) within the dose range from 25 to 750 cGy and injected with a radioprotector. Animals were subjected to a single exposure. The protective effect of β-mercaptoethylamine was tested in the experiment. The radioprotector (0.2 ml) was introduced intraperitoneally 30 minutes before exposure in 350 mI/kg dose. Control animals received the same amount of sodium chloride solution. The animals were sacrificed by cervical dislocation in 24 and 72 hrs. after exposure. It was shown that cysteamine effectively protects in vivo corneal epithelium cells of mice exposed to electromagnetic radiation or protons in a broad energy spectrum (10 to 645 MeV), and to a broad range of radiation doses (25 to 750 cGy), as judged from levels of aberrant mitosis and mitotic activity. The radioprotector exhibited the highest effectiveness in animals exposed to the doses of 50 to 300 cGy. These findings prove that cysteamine may potentially be used for pharmacological protection from protons. The radioprotector failed to prevent chromosomal aberrations after exposure to heavy charged particles of boron, carbon and neon, which implies the need to design radioprotectors against this type of corpuscular radiation specifically.

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

    PubMed

    Bath, Chris

    2013-06-01

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

  12. Corneal dystrophies

    PubMed Central

    Klintworth, Gordon K

    2009-01-01

    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 diseases (mucopolysaccharidoses

  13. A portable microkeratome for harvesting the human anterior corneal surface.

    PubMed

    Sarayba, Melvin A; Tungsiripat, Tulaya; Sweet, Paula M; Chuck, Roy S

    2004-07-01

    A portable, gas-driven turbine microkeratome device capable of harvesting the entire anterior corneal surface for lamellar transplantation on human donor globes was evaluated. The device consisted of a modified LASIK microkeratome with an enlarged suction ring, head, and blade. Vacuum was achieved by a simple hand pump. Lamellar keratectomy was performed on 5 fresh human donor globes. Lenticule dimensions were measured on days 0, 3, 6, and 9 after storage in preservation media at 4 degrees C. On day 0, the obtained lenticules were 13.9 +/- 0.9 mm and 13.5 +/- 0.4 mm, vertical and horizontal diameters, respectively. The average central lenticule thickness was 152.2 +/- 52 microm. Each lenticule was uniform in thickness over 5 measurement points (P = 0.74). Repeat measurements of corneal thickness over the 9 days showed no statistically significant difference (P = 0.51). On day 9 lenticules were 14.6 +/- 0.3 mm and 14.6 +/- 0.4 mm, vertical and horizontal diameters, respectively. When day 0 was compared to day 9, vertical diameter also showed no statistically significant difference (P = 0.16), whereas horizontal diameter was significantly different (P < 0.001). This device proves to be an economical alternative to electric-powered systems for the harvest of transplantable corneal sections.

  14. Pseudomonas aeruginosa binds to extracellular matrix deposited by human corneal epithelial cells.

    PubMed

    Esco, Miechia A; Hazlett, Linda D; Kurpakus-Wheater, Michelle

    2002-12-01

    To measure the effect of extracellular matrix substrate, pH, and O(2) on Pseudomonas aeruginosa binding. Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O(2). P. aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O(2). The mean number of adherent bacteria per counted per field +/- SEM (n = 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O(2). Binding in the presence of antibodies directed against laminin-5 was also measured. Extracellular matrix substrates produced by cells cultured in 20% O(2), combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660. In contrast, extracellular matrix substrates produced by cells cultured in 2% O(2), combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660. Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660. Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control. The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P. aeruginosa. Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix. The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion.

  15. One-year Outcomes of Pachymetry and Epithelium Thicknesses after Accelerated (45 mW/cm2) Transepithelial Corneal Collagen Cross-linking for Keratoconus Patients

    PubMed Central

    Zhang, Xiaoyu; Sun, Ling; Chen, Yingjun; Li, Meiyan; Tian, Mi; Zhou, Xingtao

    2016-01-01

    The thickness of corneal pachymetry and the epithelium after accelerated (45 mW/cm2) transepithelial corneal collagen cross-linking (CXL) for keratoconus were assessed in this prospective case series study. Twenty-eight patients were treated for keratoconus. The mean Kmax was 56.18 ± 7.90. The thinnest point, as assessed by optical coherence tomography (OCT), was 443.18 ± 39.75 μm. Accelerated transepithelial CXL was performed, and corrected distance visual acuity (CDVA), corneal topography, and OCT were recorded at 1 week postoperatively as well as at 1, 3, 6, and 12 months. The surgery was uneventful in all eyes. Postoperative epithelial edema was observed and faded in 3 days. The postoperative Kmax was 54.56 ± 8.81, 55.78 ± 8.11, 56.37 ± 8.71, 55.80 ± 7.92, and 55.47 ± 8.24 at 1 week, 1 month, 3 months, 6 months, and 12 months, respectively (all, P > 0.05). The thinnest postoperative corneal point, 439.04 ± 44.99 μm, was observed at 12 months (P = 0.109). The epithelial thickness decreased during the first postoperative week then showed a gradual recovery. Postoperative pachymetry thickness showed no significant changes for up to 12 months. Postoperative epithelial thickness decreased temporarily, then stabilized at month 12. Accelerated transepithelial CXL was shown to be effective and safe for the treatment of keratoconus. PMID:27597655

  16. Mucin-like glycoprotein secretion is mediated by cyclic-AMP and protein kinase C signal transduction pathways in rat corneal epithelium.

    PubMed

    Nakamura, M; Endo, K; Nakata, K

    1998-05-01

    Ocular surface mucin is secreted from both goblet cells in the conjunctival epithelium and corneal epithelial cells. To clarify its mechanism of secretion in corneal epithelial cells, a rat cornea organ culture system was used to evaluate the second messenger roles of cyclic-AMP (cAMP), cyclic-GMP (cGMP) and protein kinase C (PKC) in modulating mucin-like glycoprotein secretion. Rat cornea sections (3 mm diameter) were cultured in TC-199 medium, and radiolabeled with sodium sulfate for 18 hr. After washing, the corneas were treated with various second messenger modulating agents for 30 min. The culture media were reacted with Dolichos biflorus (DBA)-lectin, and mucin-like glycoprotein was isolated. Then the radioactivity of DBA-binding mucin-like glycoprotein was isolated. Then the radioactivity of DBA-binding mucin-like glycoprotein was measured. There was a time-dependent increase in mucin-like glycoprotein was measured. There was a time-dependent increase in mucin-like glycoprotein secretion, whereas after corneal epithelial debridement the secretion was markedly inhibited by 81%. Mucin-like glycoprotein secretion was stimulated in a dose-dependent manner following elevation of cAMP levels by exposure to either forskolin, dibutyryl cAMP or 3-isobutyl-1-methylxanthine. Concomitant exposure to the cAMP dependent protein kinase inhibitor, KT5720 completely inhibited their stimulatory effects. Neither exposure to dibutyryl cGMP nor nitroprusside affected mucin-like glycoprotein secretion. Stimulation by PKC, phorbol 12, 13-dibutyrate (PDBu) also increased mucin-like glycoprotein secretion in a dose-dependent fashion. The PKC inhibitor, calphostin C completely inhibited the stimulation by PDBu of mucine-like glycoprotein secretion. These results demonstrate that corneal epithelial cells secrete mucin-like glycoprotein, which is mediated by cAMP and PKC signal transduction pathways.

  17. Megalin and cubilin in the human gallbladder epithelium.

    PubMed

    Tsaroucha, Alexandra K; Chatzaki, Ekaterini; Lambropoulou, Maria; Despoudi, Kaliopi; Laftsidis, Prodromos; Charsou, Chara; Polychronidis, Alexandros; Papadopoulos, Nikolaos; Simopoulos, Constantinos E

    2008-09-01

    Although the role of cholesterol absorption by the gallbladder epithelium in gallstone formation is well established, the exact process is poorly understood. Potential candidates for regulation of transepithelial cholesterol transport are suggested to be two large membrane multiple ligand receptors, megalin and cubilin. We studied the expression of these two proteins in both acalculous and calculous human gallbladder epithelia. Adult human gallbladder tissues were received from 21 patients (9 men, 12 women) who had undergone cholecystectomy. The patients were divided into two groups: group A (calculous gallbladder group; 5 men, 6 women; mean age 64.4 +/- 11.1 years) with cholelithiasis, and group B (acalculous gallbladder group; 4 men, 6 women; mean age 55.3 +/- 16.1 years). In the gallbladder tissues megalin and cubilin expression was studied by immunohistochemistry and conventional RT-PCR, and gene expression levels were estimated by real-time RT-PCR. Both megalin and cubilin gene transcripts were found in total RNA preparations from acalculous gallbladder. In contrast, in preparations from calculous gallbladder, none or only one of the proteins was detected. Immunoreactive proteins were detected in the simple columnar acalculous gallbladder epithelium but not in the calculous gallbladder epithelium. Our results show different expression patterns of the two proteins in calculous gallbladders and acalculous gallbladders. In the latter both proteins are expressed, suggesting an association with gallstone formation and implying a putative role of the two proteins in cholesterol endocytosis. In other words, the presence of both proteins may be essential for the prevention of stone formation.

  18. Ultracytochemical study on the permeability of the human amniotic epithelium.

    PubMed

    Matsubara, S; Tamada, T

    1991-06-01

    In order to elucidate and characterize the transport pathway of the substances in the amniotic fluid, the permeability of the term human amnion was studied ultracytochemically, with lanthanum or horse radish peroxidase (HRP) as a tracer. Pieces of the term human amnion were exposed to the solutions containing lanthanum or HRP, and processed for electronmicroscopy. Precipitates indicating lanthanum or HRP were observed in the lateral intercellular spaces of the amniotic epithelial cells through the entire depth of the spaces. Generally, pinocytosis of HPR was not observed. In rare cases, however, diffuse uptake of HRP was noticed in the cells of the electron-lucent cytoplasm. These facts indicated that the human amniotic epithelium is quite permeable and that this particular intercellular pathway is important in the mechanism of the transfer of substances between the mother and the fetus.

  19. 3D map of the human corneal endothelial cell

    PubMed Central

    He, Zhiguo; Forest, Fabien; Gain, Philippe; Rageade, Damien; Bernard, Aurélien; Acquart, Sophie; Peoc’h, Michel; Defoe, Dennis M.; Thuret, Gilles

    2016-01-01

    Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions. PMID:27381832

  20. Vitamin D Induces Global Gene Transcription in Human Corneal Epithelial Cells: Implications for Corneal Inflammation.

    PubMed

    Reins, Rose Y; Mesmar, Fahmi; Williams, Cecilia; McDermott, Alison M

    2016-05-01

    Our previous studies show that human corneal epithelial cells (HCEC) have a functional vitamin D receptor (VDR) and respond to vitamin D by dampening TLR-induced inflammation. Here, we further examined the timing of the cytokine response to combined vitamin D-TLR treatment and used genome-wide microarray analysis to examine the effect of vitamin D on corneal gene expression. Telomerase-immortalized HCEC (hTCEpi) were stimulated with polyinosinic-polycytidylic acid (poly[I:C]) and 1,25-dihydroxyvitamin D3 (1,25D3) for 2 to 24 hours and interleukin (IL)-8 expression was examined by quantitative (q)PCR and ELISA. Telomerase-immortalized HCEC and SV40-HCEC were treated with 1,25D3 and used in genome-wide microarray analysis. Expression of target genes was validated using qPCR in both cell lines and primary HCEC. For confirmation of IκBα protein, hTCEpi were treated with 1,25D3 for 24 hours and cell lysates used in an ELISA. Treatment with 1,25D3 increased poly(I:C)-induced IL-8 mRNA and protein expression after 2 to 6 hours. However, when cells were pretreated with 1,25D3 for 24 hours, 1,25D3 decreased cytokine expression. For microarray analysis, 308 genes were differentially expressed by 1,25D3 treatment in hTCEpi, and 69 genes in SV40s. Quantitative (q)PCR confirmed the vitamin D-mediated upregulation of target genes, including nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα). In addition to increased transcript levels, IκBα protein was increased by 28% following 24 hours of vitamin D treatment. Microarray analysis demonstrates that vitamin D regulates numerous genes in HCEC and influences TLR signaling through upregulation of IκBα. These findings are important in dissecting the role of vitamin D at the ocular surface and highlight the need for further research into the functions of vitamin D and its influence on corneal gene expression.

  1. Proteomic analysis of the soluble fraction from human corneal fibroblasts with reference to ocular transparency.

    PubMed

    Karring, Henrik; Thøgersen, Ida B; Klintworth, Gordon K; Enghild, Jan J; Møller-Pedersen, Torben

    2004-07-01

    The transparent corneal stroma contains a population of corneal fibroblasts termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the keratocytes are quiescent and transparent. However, after corneal injury the keratocytes become activated and transform into backscattering wound-healing fibroblasts resulting in corneal opacification. At present, the most popular hypothesis suggests that particular abundant water-soluble proteins called enzyme-crystallins are involved in maintaining corneal cellular transparency. Specifically, corneal haze development is thought to be related to low levels of cytoplasmic enzyme-crystallins in reflective corneal fibroblasts. To further investigate this hypothesis, we have used a proteomic approach to identify the most abundant water-soluble proteins in serum-cultured human corneal fibroblasts that represent an in vitro model of the reflective wound-healing keratocyte phenotype. Densitometry of one-dimensional gels revealed that no single protein isoform exceeded 5% of the total water-soluble protein fraction, which is the qualifying property of a corneal enzyme-crystallin according to the current definition. This result indicates that wound-healing corneal fibroblasts do not contain enzyme-crystallins. A total of 254 protein identifications from two-dimensional gels were performed representing 118 distinct proteins. Proteins protecting against oxidative stress and protein misfolding were prominent, suggesting that these processes may participate in the generation of cytoplasmic light-scattering from corneal fibroblasts.

  2. Aloe vera extract activity on human corneal cells.

    PubMed

    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.

  3. Effect of Streptococcus pneumoniae on human respiratory epithelium in vitro.

    PubMed

    Steinfort, C; Wilson, R; Mitchell, T; Feldman, C; Rutman, A; Todd, H; Sykes, D; Walker, J; Saunders, K; Andrew, P W

    1989-07-01

    A total of 11 of 15 Streptococcus pneumoniae culture filtrates and all five bacterial autolysates produced by cell death in the stationary phase caused slowed ciliary beating and disruption of the surface integrity of human respiratory epithelium in organ culture. This effect was inhibited by cholesterol and was heat labile and reduced by standing at room temperature but was stable at -40 degrees C. The activity was detected at the late stationary phase of culture and was associated with the presence of hemolytic activity. Gel filtration of a concentrated culture filtrate and autolysate both yielded a single fraction of approximately 50 kilodaltons which slowed ciliary beating and were the only fractions with hemolytic activity. Rabbit antiserum to pneumolysin, a sulfhydryl-activated hemolytic cytotoxin released by S. pneumoniae during autolysis, neutralized the effect of the culture filtrate on respiratory epithelium. Both native and recombinant pneumolysin caused ciliary slowing and epithelial disruption. Electron microscopy showed a toxic effect of pneumolysin on epithelial cells: cytoplasmic blebs, mitochondrial swelling, cellular extrusion, and cell death, but no change in ciliary ultrastructure. Recombinant pneumolysin (10 micrograms/ml) caused ciliary slowing in the absence of changes in cell ultrastructure. Release of pneumolysin in the respiratory tract during infection may perturb host defenses, allowing bacterial proliferation and spread.

  4. An Apical-Membrane Chloride Channel in Human Tracheal Epithelium

    NASA Astrophysics Data System (ADS)

    Welsh, Michael J.

    1986-06-01

    The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.

  5. Effect of nitrogen dioxide on human nasal epithelium

    SciTech Connect

    Carson, J.L.; Collier, A.M.; Hu, S.C.; Delvin, R.B. )

    1993-09-01

    The nasal epithelium of young adult white men in good health was evaluated by electron microscopy in a condition blind fashion relative to exposures of 2 ppm nitrogen dioxide (NO2) or clean air for 4 h. The exposure protocol involved two separate exposures of the same individuals to NO2 or clean air approximately 3 wk apart. We found qualitative and quantitative evidence that luminal border membranes of ciliated cells were ultrastructurally altered in six of seven samples of nasal epithelium obtained following NO2 exposures, although subsequent morphometric statistical analyses were not significant. This alteration was characterized by cilia containing excess matrix in which individual or, more commonly, multiple ciliary axonemes were embedded, and by vesiculations of luminal border ciliary membranes, a pattern less common in clean air-exposed control specimens. Although these patterns were not widespread, their morphology was consistent with findings of previous animal studies involving acute and chronic exposure to NO2. Our findings suggest that adverse effects on mucociliary function in normal humans due to acute exposure to low levels of NO2 are most likely minimal. However, in view of other reports of NO2 exposure in laboratory animals documenting ciliary injury, our observations support a view that similar patterns might appear more prominently with higher NO2 levels and/or more extended exposure intervals.

  6. Two percent ethylenediaminetetraacetic acid chelation treatment for band-shaped keratopathy, without blunt scratching after removal of the corneal epithelium

    PubMed Central

    Kobayashi, Wataru; Yokokura, Shunji; Hariya, Takehiro; Nakazawa, Toru

    2015-01-01

    Background The purpose of this study was to assess the effectiveness of 2% ethylenediaminetetraacetic acid (EDTA) for the treatment of band-shaped keratopathy. Methods We studied 24 eyes of 16 patients with band-shaped keratopathy who underwent EDTA chelation treatment from April 1, 2011 to December 31, 2012. We compared preoperative and 1 month postoperative logarithm of the minimum angle of resolution (logMAR) best corrected visual acuity, intraocular pressure, and corneal curvature radius (K1, horizontal meridian; K2, vertical meridian; Km, average of K1 and K2). The Mann-Whitney U-test was used to determine the significance of differences. Results There was a significant difference in preoperative and postoperative logMAR best corrected visual acuity (P=0.01). There were no significant differences in preoperative and postoperative intraocular pressure (P=0.24) or corneal curvature radius (K1, P=0.54; K2, P=0.49; Km, P=0.45). Conclusion After 2% EDTA chelation treatment, post-operative logMAR best corrected visual acuity improved significantly. Moreover, since there was no significant difference in corneal curvature radius, there was little influence on corneal surface form. We believe that the results of our 2% EDTA chelation treatment were comparable with results obtained with 3.75% EDTA chelation treatment in previous reports. Two percent EDTA chelation is an effective treatment for band-shaped keratopathy and a useful method for any institution. PMID:25673973

  7. Effect of Schizandra chinensis lignans on cell division in the corneal epithelium and tongue of albino rats exposed to chronic cold stress

    SciTech Connect

    Mel'nik, E.I.; Lupandin, A.V.; Timoshin, S.S.

    1985-05-01

    The authors study the possibility of correcting cellular manifestations of disadaptation following chronic exposure to cold stress by means of preparations of Sch. chinensis. The model of chronic stress was cooling male albino rats daily for 1.5 h to a temperature of 28-30 C for 28 days. Since differences between levels of proliferation in intact animals and in the rats receiving 1.9% ethanol solution were absent, values obtained in the group of intact animals are presented in a table as the control. The animals underwent euthanasia 48 hours after the final exposure to the cold. The rats received an injection of tritium-thymidine one hour before sacrifice. It is shown that the results confirm those in previous studies of stimulation of DNA synthesis and mitotic activity in the corneal and lingual epithelium of albino rats during chronic exposure to stress.

  8. Bilateral lesions of suprachiasmatic nuclei affect circadian rhythms in (/sup 3/H)-thymidine incorporation into deoxyribonucleic acid in mouse intestinal tract, mitotic index of corneal epithelium, and serum corticosterone

    SciTech Connect

    Scheving, L.E.; Tsai, T.H.; Powell, E.W.; Pasley, J.N.; Halberg, F.; Dunn, J.

    1983-03-01

    Investigations into the role of the suprachiasmatic nuclei (SCN) in the coordination of circadian rhythms have presented differing results. Several reports have shown that ablation of the suprachiasmatic nuclei (SCNA) alters the phase and amplitude of rhythms but does not abolish them. The present study investigates the effect of SCNA on the rhythms in cell proliferation in various regions of the intestinal tract as measured by the incorporation of (/sup 3/H)-thymidine into deoxyribonucleic acid, in the mitotic activity of the corneal epithelium, and in serum corticosterone levels. The study involved mice with verified lesions of the SCN (six to 13 mice per time point) and control groups of both sham-operated and unoperated mice (seven of each per time point). The mice were killed in groups that represented seven time points over a single 24 hr span (3 hr intervals with the 0800 hr sampled both at start and end of the series). The tissues examined were the tongue, esophagus, gastric stomach, and colon for DNA synthesis, the corneal epithelium for mitotic index, and blood serum for corticosterone level. The most consistent result of SCNA was a phase advance in the rhythms in cell proliferation in the tongue, esophagus, gastric stomach, colon, and corneal epithelium. A reduction in rhythm amplitude occurred in the tongue, esophagus, and corneal epithelium; however, there was an amplitude increase for the stomach, colon, and serum corticosterone. The mesor (rhythm-adjusted mean) was increased by SCNA in all tissues except the corneal epithelium. These findings further support the role of the suprachiasmatic nuclear area in the control of rhythms in cell proliferation and corticosterone production, by acting as a ''phase-resetter'' and as a modulator of rhythm amplitude.

  9. Regulation of human corneal epithelial mucins by rebamipide.

    PubMed

    Itoh, Shinsaku; Itoh, Kuni; Shinohara, Hisashi

    2014-02-01

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

  10. Transcriptome of the human retina, retinal pigmented epithelium and choroid

    PubMed Central

    Tian, Lifeng; Kazmierkiewicz, Krista L; Bowman, Anita S; Li, Mingyao; Curcio, Christine A; Stambolian, Dwight E

    2015-01-01

    The retina and its adjacent supporting tissues -- retinal pigmented epithelium (RPE) and choroid -- are critical structures in human eyes required for normal visual perception. Abnormal changes in these layers have been implicated in diseases such as age-related macular degeneration and glaucoma. With the advent of high-throughput methods, such as serial analysis of gene expression, cDNA microarray, and RNA sequencing, there is unprecedented opportunity to facilitate our understanding of the normal retina, RPE, and choroid. This information can be used to identify dysfunction in age-related macular degeneration and glaucoma. In this review, we describe the current status in our understanding of these transcriptomes through the use of high throughput techniques. PMID:25645700

  11. Decellularization of human stromal refractive lenticules for corneal tissue engineering

    PubMed Central

    Yam, Gary Hin-Fai; Yusoff, Nur Zahirah Binte M.; Goh, Tze-Wei; Setiawan, Melina; Lee, Xiao-Wen; Liu, Yu-Chi; Mehta, Jodhbir S.

    2016-01-01

    Small incision lenticule extraction (SMILE) becomes a procedure to correct myopia. The extracted lenticule can be used for other clinical scenarios. To prepare for allogeneic implantation, lenticule decellularization with preserved optical property, stromal architecture and chemistry would be necessary. We evaluated different methods to decellularize thin human corneal stromal lenticules created by femtosecond laser. Treatment with 0.1% sodium dodecylsulfate (SDS) followed by extensive washes was the most efficient protocol to remove cellular and nuclear materials. Empty cell space was found inside the stroma, which displayed aligned collagen fibril architecture similar to native stroma. The SDS-based method was superior to other treatments with hyperosmotic 1.5 M sodium chloride, 0.1% Triton X-100 and nucleases (from 2 to 10 U/ml DNase and RNase) in preserving extracellular matrix content (collagens, glycoproteins and glycosaminoglycans). The stromal transparency and light transmittance was indifferent to untreated lenticules. In vitro recellularization showed that the SDS-treated lenticules supported corneal stromal fibroblast growth. In vivo re-implantation into a rabbit stromal pocket further revealed the safety and biocompatibility of SDS-decellularized lenticules without short- and long-term rejection risk. Our results concluded that femtosecond laser-derived human stromal lenticules decellularized by 0.1% SDS could generate a transplantable bioscaffold with native-like stromal architecture and chemistry. PMID:27210519

  12. Human milk hyaluronan enhances innate defense of the intestinal epithelium.

    PubMed

    Hill, David R; Rho, Hyunjin K; Kessler, Sean P; Amin, Ripal; Homer, Craig R; McDonald, Christine; Cowman, Mary K; de la Motte, Carol A

    2013-10-04

    Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.

  13. Human Milk Hyaluronan Enhances Innate Defense of the Intestinal Epithelium*

    PubMed Central

    Hill, David R.; Rho, Hyunjin K.; Kessler, Sean P.; Amin, Ripal; Homer, Craig R.; McDonald, Christine; Cowman, Mary K.; de la Motte, Carol A.

    2013-01-01

    Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn. PMID:23950179

  14. Acid phosphatase and lipid peroxidation in human cataractous lens epithelium.

    PubMed

    Vasavada, A R; Thampi, P; Yadav, S; Rawal, U M

    1993-12-01

    The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium) and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium). In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium) and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium). From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.

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

    PubMed

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

    2014-06-01

    To evaluate an automated process to find borders of corneal basal epithelial cells in pictures obtained from in vivo laser scanning confocal microscopy (Heidelberg Retina Tomograph III with Rostock corneal module). On a sample of 20 normal corneal epithelial pictures, images were segmented through an automated four-step segmentation algorithm. Steps of the algorithm included noise reduction through a fast Fourier transform (FFT) band-pass filter, image binarization with a mean value threshold, watershed segmentation algorithm on distance map to separate fused cells and Voronoi diagram segmentation algorithm (which gives a final mask of cell borders). Cells were then automatically counted using this border mask. On the original image either with contrast enhancement or noise reduction, cells were manually counted by a trained operator. The average cell density was 7722.5 cells/mm(2) as assessed by automated analysis and 7732.5 cells/mm(2) as assessed by manual analysis (p = 0.93). Correlation between automated and manual analysis was strong (r = 0.974 [0.934-0.990], p < 0.001). Bland-Altman method gives a mean difference in density of 10 cells/mm(2) and a limits of agreement ranging from -971 to +991 cells/mm(2) . Visually, the algorithm correctly found almost all borders. This automated segmentation algorithm is worth for assessing corneal epithelial basal cell density and morphometry. This procedure is fully reproducible, with no operator-induced variability. © 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  16. Human Bone Marrow Stromal Cells Differentiate Into Corneal Tissue and Prevent Ocular Graft-Versus-Host Disease in Mice.

    PubMed

    Sánchez-Abarca, Luis Ignacio; Hernández-Galilea, Emiliano; Lorenzo, Rebeca; Herrero, Carmen; Velasco, Almudena; Carrancio, Soraya; Caballero-Velázquez, Teresa; Rodríguez-Barbosa, José Ignacio; Parrilla, Marta; Del Cañizo, Consuelo; San Miguel, Jesús; Aijón, José; Pérez-Simón, José Antonio

    2015-01-01

    Clinical trials have assessed the use of human bone marrow stromal cells (hBMSCs) for the treatment of immune-related disorders such as graft-versus-host disease (GVHD). In the current study, we show that GFP(+)-transduced hBMSCs generated from bone marrow migrate and differentiate into corneal tissue after subconjunctival injection in mice. Interestingly, these hBMSCs display morphological features of epithelial, stromal, and endothelial cells and appear at different layers and with different morphologies depending on their position within the epithelium. Furthermore, these cells display ultrastructural properties, such as bundles of intermediate filaments, interdigitations, and desmosomes with GFP(-) cells, which confirms their differentiation into corneal tissues. GFP(+)-transduced hBMSCs were injected at different time points into the right eye of lethally irradiated mice undergoing bone marrow transplantation, which developed ocular GVHD (oGVHD). Remarkably, hBMSCs massively migrate to corneal tissues after subconjunctival injection. Both macroscopic and histopathological examination showed minimal or no evidence of GVHD in the right eye, while the left eye, where no hBMSCs were injected, displayed features of GVHD. Thus, in the current study, we confirm that hBMSCs may induce their therapeutic effect at least in part by differentiation and regeneration of damaged tissues in the host. Our results provide experimental evidence that hBMSCs represent a potential cellular therapy to attenuate oGVHD.

  17. Human corneal equivalent as cell culture model for in vitro drug permeation studies

    PubMed Central

    Reichl, S; Bednarz, J; Müller-Goymann, C C

    2004-01-01

    Aims: For the study of transcorneal in vitro permeation of ophthalmic drugs, excised animal cornea or corneal epithelial cell culture are frequently used as a replacement for the human cornea. The main purposes of this study were to reconstruct a complete human organotypic cornea equivalent, consisting of all three different cell types (epithelial, stromal, and endothelial); to test the barrier function of this bio-engineered human cornea using three different model drugs (pilocarpine hydrochloride (PHCl), befunolol hydrochloride (BHCl), and hydrocortisone (HC)); and to determine its usefulness as an in vitro model for prediction of ocular drug absorption into the human eye. Methods: A multilayer tissue construct was created step by step in Transwell cell culture insert using SV-40 immortalised human endothelial and epithelial cells and native stromal cells (fibroblasts). Morphology was characterised by light microscopy using routine H&E staining. Scanning electron microscopy was used to evaluate ultrastructural features. Ocular permeation of drugs across the human cornea construct was tested using modified Franz cells and compared with data obtained from excised porcine cornea and previously described porcine cornea constructs. Results and conclusion: The cornea construct exhibited typical corneal structures such as a monolayer of hexagonally shaped endothelial cells and a multilayered epithelium consisting of seven to nine cell layers with flat superficial cells. The formation of microplicae and microvilli was also confirmed. The human cornea construct showed similar permeation behaviour for all substances compared with excised porcine cornea. However, permeability (permeation coefficients Kp) of the human cornea equivalent (PHCl 13.4•10−6 (SD 3.01•10−6); BHCl 9.88•10−6 (SD 1.79•10−6); HC 5.41•10−6 (SD 0.40•10−6) cm/s) was about 1.6–1.8 fold higher than excised porcine cornea. Compared with data from the porcine cornea construct the

  18. Somatic Variants in the Human Lens Epithelium: A Preliminary Assessment

    PubMed Central

    Mesa, Rosana; Tyagi, Manoj; Harocopos, George; Vollman, David; Bassnett, Steven

    2016-01-01

    Purpose We hypothesize that somatic mutations accumulate in cells of the human lens and may contribute to the development of cortical or posterior sub-capsular cataracts. Here, we used a Next-generation sequencing (NGS) strategy to screen for low-allelic frequency variants in DNA extracted from human lens epithelial samples. Methods Next-Generation sequencing of 151 cancer-related genes (WUCaMP2 panel) was performed on DNA extracted from post-mortem or surgical specimens obtained from 24 individuals. Usually, pairwise comparisons were made between two or more ocular samples from the same individual, allowing putative somatic variants detected in lens samples to be differentiated from germline variants. Results Use of a targeted hybridization approach enabled high sequence coverage (>1000-fold) of the WUCaMP2 genes. In addition to high-frequency variants (corresponding to homozygous or heterozygous SNPs and Indels), somatic variants with allelic frequencies of 1-4% were detected in the lens epithelial samples. The presence of one such variant, a T > C point substitution at position 32907082 in BRCA2, was verified subsequently using droplet digital PCR. Conclusions Low-allelic fraction variants are present in the human lens epithelium, at frequencies consistent with the presence of millimeter-sized clones. PMID:27537255

  19. Rapid tissue engineering of biomimetic human corneal limbal crypts with 3D niche architecture.

    PubMed

    Levis, Hannah J; Massie, Isobel; Dziasko, Marc A; Kaasi, Andreas; Daniels, Julie T

    2013-11-01

    Limbal epithelial stem cells are responsible for the maintenance of the human corneal epithelium and these cells reside in a specialised stem cell niche. They are located at the base of limbal crypts, in a physically protected microenvironment in close proximity to a variety of neighbouring niche cells. Design and recreation of elements of various stem cell niches have allowed researchers to simplify aspects of these complex microenvironments for further study in vitro. We have developed a method to rapidly and reproducibly create bioengineered limbal crypts (BLCs) in a collagen construct using a simple one-step method. Liquid is removed from collagen hydrogels using hydrophilic porous absorbers (HPAs) that have custom moulded micro-ridges on the base. The resulting topography on the surface of the thin collagen constructs resembles the dimensions of the stromal crypts of the human limbus. Human limbal epithelial cells seeded onto the surface of the constructs populate these BLCs and form numerous layers with a high proportion of the cells lining the crypts expressing putative stem cell marker, p63α. The HPAs are produced using a moulding process that is flexible and can be adapted depending on the requirements of the end user. Creation of defined topographical features using this process could be applicable to numerous tissue-engineering applications where varied 3-dimensional niche architectures are required.

  20. The structural and optical properties of type III human collagen biosynthetic corneal substitutes

    PubMed Central

    Hayes, Sally; Lewis, Phillip; Islam, M. Mirazul; Doutch, James; Sorensen, Thomas; White, Tomas; Griffith, May; Meek, Keith M.

    2015-01-01

    The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2–9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants. PMID:26159106

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. Generation of Corneal Keratocytes from Human Embryonic Stem Cells

    PubMed Central

    Hertsenberg, Andrew J.; Funderburgh, James L.

    2017-01-01

    Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype. PMID:26026882

  4. "All-laser" endothelial corneal transplant in human patients

    NASA Astrophysics Data System (ADS)

    Rossi, Francesca; Menabuoni, Luca; Malandrini, Alex; Canovetti, Annalisa; Lenzetti, Ivo; Pini, Roberto

    2012-03-01

    Femtosecond laser sculpturing of corneal tissue is commonly used for the preparation of endothelial flaps. Diode laser welding of ocular tissues is a procedure that enables minimally invasive suturing of tissues. The combination of these laser based techniques results in a new approach to minimally invasive ophthalmic surgery, such as in endothelial corneal transplant (or endothelial keratoplasty - EK). In this work we present the "all laser" EK performed in human subjects. 24 pseudophakic patients with bullous keratopathy underwent EK: the femtosecond laser was used to prepare the 100 ìm thick and 8.5 mm diameter donor Descemet endothelial flap. After staining the stromal layer of the donor flap with a liquid ICG solution, the donor flap was inserted in the recipient eye by the use of the Busin injector. Then, the endothelial layer was laser-welded to the recipient eye (10 laser spots around the periphery of the flap), in order to reduce the risk of postoperative dislocation of the transplanted flap. A transplanted flap engraftment was observed in all the treated eyes. The staining procedure used to perform laser welding also enabled to evidence the stromal side of the donor flap, so as the flap was always placed in the right side position. The endothelial cells counts in both the laserwelded flaps and in a control group were in good agreement. The proposed technique is easy to perform and enables the reduction of postoperative endothelial flap dislocations.

  5. Cytotoxicity testing of multipurpose contact lens solutions using monolayer and stratified cultures of human corneal epithelial cells.

    PubMed

    Lim, Mary J; Hurst, Rachel K; Konynenbelt, B J; Ubels, John L

    2009-11-01

    To investigate in vitro cytotoxicity of five marketed multipurpose contact lens solutions (MPS) on the morphology, viability, and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Cells were exposed to MPS for 10, 20, or 60 minutes. In monolayer cultures, effects of the MPS on cell morphology were observed using Hoffman modulation contrast microscopy. Cell proliferation after exposure to MPS was evaluated and cytotoxicity of the MPS was determined using a live/dead cell assay and flow cytometry. In stratified cultures, multilayer corneal epithelial constructs were established on membrane inserts. Effects of the MPS on the morphology and barrier function of stratified cultures were determined using microscopy, a fluorescein permeability test, and measurement of trans-epithelial resistance. In monolayer cultures, none of the MPS damaged cells during a 10-min exposure. All of the MPS had varying time-dependent adverse effects on cell morphology, viability, and proliferation during 20- and 60-min exposures. In stratified cultures, none of the MPS had an adverse effect on the structure or barrier function of stratified cultures. Monolayer cultures are highly sensitive to damage by MPS. In contrast, because stratified human corneal-limbal epithelial cultures are resistant to adverse effects of MPS, it is suggested that models that simulate the stratified structure of the corneal epithelium should be used for in vitro toxicologic testing. Caution should be used when interpreting such studies, because in vitro tests may not be predictive of clinical responses to contact lens products that are known to be safe when used as directed.

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

    PubMed Central

    McCabe, Kathryn L.; Kunzevitzky, Noelia J.; Chiswell, Brian P.; Xia, Xin; Goldberg, Jeffrey L.; Lanza, Robert

    2015-01-01

    Aim To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. Materials and Methods 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. Results 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. Conclusion 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. PMID:26689688

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

    NASA Astrophysics Data System (ADS)

    Tocce, Elizabeth J.

    2011-12-01

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

  8. TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium.

    PubMed

    Martínez-Rendón, Jacqueline; Sánchez-Guzmán, Erika; Rueda, Angélica; González, James; Gulias-Cañizo, Rosario; Aquino-Jarquín, Guillermo; Castro-Muñozledo, Federico; García-Villegas, Refugio

    2017-07-01

    TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca(2+) imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Molecular Expression and Functional Evidence of a Drug Efflux Pump (BCRP) in Human Corneal Epithelial Cells

    PubMed Central

    Karla, Pradeep K.; Earla, Ravinder; Boddu, Sagar H.; Johnston, Thomas P.; Pal, Dhananjay; Mitra, Ashim

    2015-01-01

    Purpose Breast Cancer Resistance Protein (BCRP) belongs to the 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 the presence and possible role of BCRP as a bar-rier for ocular drug resistance. Methods Transfected human corneal epithelial cells (SV40-HCEC) were selected as an in vitro model for corneal epithelium with MDCKII-BCRP as positive control. [3H]-Mitoxantrone ([3H]-MTX), which is a proven substrate for organic anion transporter like BCRP, was selected as a model drug for functional expression studies. Fumetremorgin C (FTC), a known specific inhibitor for BCRP and GF120918, an inhibitor for BCRP and P-gp, were added to inhibit BCRP-mediated efflux. PGP-4008, a specific inhibitor of P-gp was used to delineate the contribution of P-gp. The mRNA extracted from cells was used for RT-PCR analysis and gene expression. Membrane fractions of SV40-HCEC and MDCKII-BCRP were used for immunoprecipitation followed by Western blot analysis. Results Efflux was inhibited significantly in the presence of FTC and GF120918. Dose-dependent inhibition of efflux by BCRP was noticed in SV40-HCEC and MDCKII-BCRP in the presence of FTC and GF120918, and the efflux was ATP-dependent. The metabolic inhibitor, 2,4-DNP, significantly inhibited efflux. No pH-dependent efflux was noticed except at pH 5.5. RT-PCR analysis indicated a unique and distinct band at ~429 bp, corresponding to BCRP in SV40-HCEC and MDCKII-BCRP cells. Western Blot analysis indicated a specific band at ~70 kDa in the membrane fraction of SV40-HCEC and MDCKII-BCRP cells. Conclusions We have demonstrated the expression of BCRP in human corneal epithelial cells and, for the first time, demonstrated its functional activity leading to drug efflux. RT-PCR and Western blot analysis further confirmed this finding. PMID:19172464

  10. Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye.

    PubMed

    Argüeso, Pablo; Tisdale, Ann; Spurr-Michaud, Sandra; Sumiyoshi, Mika; Gipson, Ilene K

    2006-01-01

    Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake

  11. Role of Human Corneal Stroma-Derived Mesenchymal-Like Stem Cells in Corneal Immunity and Wound Healing

    PubMed Central

    Veréb, Zoltán; Póliska, Szilárd; Albert, Réka; Olstad, Ole Kristoffer; Boratkó, Anita; Csortos, Csilla; Moe, Morten C.; Facskó, Andrea; Petrovski, Goran

    2016-01-01

    Corneal tissue regeneration is of crucial importance for maintaining normal vision. We aimed to isolate and cultivate human corneal stroma-derived mesenchymal stem-like cells (CSMSCs) from the central part of cadaver corneas and study their phenotype, multipotency, role in immunity and wound healing. The isolated cells grew as monolayers in vitro, expressed mesenchymal- and stemness-related surface markers (CD73, CD90, CD105, CD140b), and were negative for hematopoietic markers as determined by flow cytometry. CSMSCs were able to differentiate in vitro into fat, bone and cartilage. Their gene expression profile was closer to bone marrow-derived MSCs (BMMSCs) than to limbal epithelial stem cells (LESC) as determined by high-throughput screening. The immunosuppressive properties of CSMSCs were confirmed by a mixed lymphocyte reaction (MLR), while they could inhibit proliferation of activated immune cells. Treatment of CSMSCs by pro-inflammatory cytokines and toll-like receptor ligands significantly increased the secreted interleukin-6 (IL-6), interleukin-8 (IL-8) and C-X-C motif chemokine 10 (CXCL-10) levels, as well as the cell surface adhesion molecules. CSMSCs were capable of closing a wound in vitro under different stimuli. These cells thus contribute to corneal tissue homeostasis and play an immunomodulatory and regenerative role with possible implications in future cell therapies for treating sight-threatening corneal diseases. PMID:27195722

  12. Activation of human lymphocytes by supernatants from human thymic epithelium.

    PubMed

    Goust, J M; Vesole, D H; Fudenberg, H H

    1979-11-01

    Supernatants from human thymic epithelial cells (TS) were found to have a mitogenic effect on cultured human peripheral blood mononuclear cells and to potentiate their responses to lectins. This was not observed with culture supernatants from the human cell lines AV-3 and HeLa or from the murine cell line L-929. The maximum potentiating effects were observed with pokeweed mitogen (PWM) and phytohaemagglutinin (PHA), whereas the response to concanavalin A (Con A) was only slightly enhanced. TS also potentiated the mixed lymphocyte culture (MLC) response of normal T cells and thymocytes cultured with mitomycin C-treated B lymphoid cell lines. The mitogenic effect of TS was time-dependent and paralleled the appearance of lymphoid colonies in semi-solid agar. Chromatographical separation of concentrated serum-free TS on Sephadex G-100 yielded an active fraction of molecular weight 15,000--25,000 which had all the activities of unseparated TS.

  13. Activation of human lymphocytes by supernatants from human thymic epithelium.

    PubMed Central

    Goust, J M; Vesole, D H; Fudenberg, H H

    1979-01-01

    Supernatants from human thymic epithelial cells (TS) were found to have a mitogenic effect on cultured human peripheral blood mononuclear cells and to potentiate their responses to lectins. This was not observed with culture supernatants from the human cell lines AV-3 and HeLa or from the murine cell line L-929. The maximum potentiating effects were observed with pokeweed mitogen (PWM) and phytohaemagglutinin (PHA), whereas the response to concanavalin A (Con A) was only slightly enhanced. TS also potentiated the mixed lymphocyte culture (MLC) response of normal T cells and thymocytes cultured with mitomycin C-treated B lymphoid cell lines. The mitogenic effect of TS was time-dependent and paralleled the appearance of lymphoid colonies in semi-solid agar. Chromatographical separation of concentrated serum-free TS on Sephadex G-100 yielded an active fraction of molecular weight 15,000--25,000 which had all the activities of unseparated TS. PMID:160851

  14. Cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells.

    PubMed

    Suh, Leejee H; Zhang, Cheng; Chuck, Roy S; Stark, Walter J; Naylor, Stuart; Binley, Katie; Chakravarti, Shukti; Jun, Albert S

    2007-07-01

    To determine the viability and potential usefulness of cryopreserved human primary cultured corneal endothelial cells by characterizing their morphology, gene expression, and ability for genetic modification by the lentiviral vector equine infectious anemia virus (EIAV). Primary cultured endothelial cells were dissociated from human corneas and grown in organ culture medium. Corneal endothelial cell origin was confirmed by morphology and immunostaining with polyclonal anti-collagen VIII antibodies. Cells of different passages were cryopreserved in medium containing dimethyl sulfoxide and were assessed after thawing for morphology, proliferative capacity, gene expression, and ability to form cell-cell junctions. EIAV encoding enhanced green fluorescent protein (eGFP) was used to transduce cryopreserved human corneal endothelial cells. Transduced cells were then sorted by fluorescence-activated cell sorting (FACS) and imaged with fluorescence microscopy. Cryopreserved, primary, cultured human corneal endothelial cells are viable and retain their ability to proliferate, produce collagen VIII, and express ZO-1, a tight-junction protein. EIAV-based gene transfer of eGFP is highly efficient and nontoxic to cryopreserved human primary cultured corneal endothelial cells. These genetically modified cells can be selected to nearly pure populations with FACS sorting. Human primary cultured corneal endothelial cells retain their phenotypic properties after cryopreservation. The ability to store, genetically modify, and sort these cells through FACS to pure populations has the potential to greatly expand their future therapeutic application to treat corneal endothelial disorders.

  15. Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation.

    PubMed

    Parekh, Mohit; Ferrari, Stefano; Sheridan, Carl; Kaye, Stephen; Ahmad, Sajjad

    2016-02-01

    The cornea forms the front window of the eye, enabling the transmission of light to the retina through a crystalline lens. Many disorders of the cornea lead to partial or total blindness, and therefore corneal transplantation becomes mandatory. Recently, selective corneal layer (as opposed to full thickness) transplantation has become popular because this leads to earlier rehabilitation and visual outcomes. Corneal endothelial disorders are a common cause of corneal disease and transplantation. Corneal endothelial transplantation is successful but limited worldwide because of lower donor corneal supply. Alternatives to corneal tissue for endothelial transplantation therefore require immediate attention. The field of human corneal endothelial culture for transplantation is rapidly emerging as a possible viable option. This manuscript provides an update regarding these developments. Significance: The cornea is the front clear window of the eye. It needs to be kept transparent for normal vision. It is formed of various layers of which the posterior layer (the endothelium) is responsible for the transparency of the cornea because it allows the transport of ions and solutes to and from the other layers of the cornea. Corneal blindness that results from the corneal endothelial dysfunction can be treated using healthy donor tissues. There is a huge demand for human donor corneas but limited supply, and therefore there is a need to identify alternatives that would reduce this demand. Research is underway to understand the isolation techniques for corneal endothelial cells, culturing these cells in the laboratory, and finding possible options to transplant these cells in the patients. This review article is an update on the recent developments in this field.

  16. Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation

    PubMed Central

    Parekh, Mohit; Ferrari, Stefano; Sheridan, Carl; Kaye, Stephen

    2016-01-01

    The cornea forms the front window of the eye, enabling the transmission of light to the retina through a crystalline lens. Many disorders of the cornea lead to partial or total blindness, and therefore corneal transplantation becomes mandatory. Recently, selective corneal layer (as opposed to full thickness) transplantation has become popular because this leads to earlier rehabilitation and visual outcomes. Corneal endothelial disorders are a common cause of corneal disease and transplantation. Corneal endothelial transplantation is successful but limited worldwide because of lower donor corneal supply. Alternatives to corneal tissue for endothelial transplantation therefore require immediate attention. The field of human corneal endothelial culture for transplantation is rapidly emerging as a possible viable option. This manuscript provides an update regarding these developments. Significance The cornea is the front clear window of the eye. It needs to be kept transparent for normal vision. It is formed of various layers of which the posterior layer (the endothelium) is responsible for the transparency of the cornea because it allows the transport of ions and solutes to and from the other layers of the cornea. Corneal blindness that results from the corneal endothelial dysfunction can be treated using healthy donor tissues. There is a huge demand for human donor corneas but limited supply, and therefore there is a need to identify alternatives that would reduce this demand. Research is underway to understand the isolation techniques for corneal endothelial cells, culturing these cells in the laboratory, and finding possible options to transplant these cells in the patients. This review article is an update on the recent developments in this field. PMID:26702128

  17. Transcriptome analysis and molecular signature of human retinal pigment epithelium

    PubMed Central

    Strunnikova, N.V.; Maminishkis, A.; Barb, J.J.; Wang, F.; Zhi, C.; Sergeev, Y.; Chen, W.; Edwards, A.O.; Stambolian, D.; Abecasis, G.; Swaroop, A.; Munson, P.J.; Miller, S.S.

    2010-01-01

    Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed ‘signature’ genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases. PMID:20360305

  18. [Investigation of the processes of post-radiation reparation of the corneal epithelium cells of mice irradiated by helium ions with various LET values].

    PubMed

    Vorozhtsova, S V; Fedorenko, S B; Shafirkin, A V; Chikhladze, Ts A

    2008-01-01

    Biological effectiveness and post-radiation recovery of corneal epithelium cells of mice were studied. Mice were exposed to a broad range of doses from accelerated helium ions with the energy of 4 MeV/nucleon and 4 GeV/nucleon once and twice with a 24-hr. interval. LET values for these energies were 120 and 0.88 keV/microm, respectively. Gamma-source 137Cs (LET = 0.3 keV/microm) was used as a radiation standard. Animals irradiated by 25-400 sGy were sacrificed in 24 and 72 hrs. by way of cervical dislocation. Total corneal preparations made from enucleated eyeballs were analyzed for the mitotic index and aberrant mitoses using the anaphase method. High damaging properties of accelerated helium ions with the energy of 4 MeV/nucleon were manifested by a considerable reduction of the mitotic index and a more marked rise in the number of aberrant mitoses following the single and repeated exposure. Fractional irradiation by helium ions with the energy of 4 MeV/nucleon resulted in inhibition and even arrest of reparation suggesting additivity of separate radiation fractions. From the dose values that produced the double reduction of the mitotic index, coefficients of the relative biological effectiveness (RBE) for helium ions with the energy of 4 MeV/nucleon and 4 GeV/nucleon were estimated to be 5.3-8.6 and 1.3-1.8, respectively. In the context of the number of aberrant mitoses, RBE coefficients were significantly lower ranging 2.3-3.5 and 1.1-1.3 for these energies, respectively.

  19. Human Adenovirus Type 37 Uses αVβ1 and α3β1 Integrins for Infection of Human Corneal Cells

    PubMed Central

    Storm, Rickard J.; Persson, B. David; Skalman, Lars Nygård; Frängsmyr, Lars; Lindström, Mona; Rankin, Greg; Lundmark, Richard; Domellöf, Fatima Pedrosa

    2016-01-01

    ABSTRACT Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVβ3, αVβ5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVβ5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, β1, and β4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, β3, or β5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVβ1 and α3β1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and β1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs. IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVβ5 in order to enter nonocular human cells. In this study, we found that αVβ5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin

  20. LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis

    PubMed Central

    Nakamura, Takahiro; Hamuro, Junji; Takaishi, Mikiro; Simmons, Szandor; Maruyama, Kazuichi; Zaffalon, Andrea; Bentley, Adam J.; Kawasaki, Satoshi; Nagata-Takaoka, Maho; Fullwood, Nigel J.; Itami, Satoshi; Sano, Shigetoshi; Ishii, Masaru; Barrandon, Yann; Kinoshita, Shigeru

    2013-01-01

    Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1–/– mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1–/– mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow–derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis. PMID:24316976

  1. Functional annotation of the human retinal pigment epithelium transcriptome

    PubMed Central

    Booij, Judith C; van Soest, Simone; Swagemakers, Sigrid MA; Essing, Anke HW; Verkerk, Annemieke JMH; van der Spek, Peter J; Gorgels, Theo GMF; Bergen, Arthur AB

    2009-01-01

    Background To determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy human donor eyes (aged 63–78 years) were laser dissected and used for 22k microarray studies (Agilent technologies). Data were analyzed with Rosetta Resolver, the web tool DAVID and Ingenuity software. Results In total, we identified 19,746 array entries with significant expression in the RPE. Gene expression was analyzed according to expression levels, interindividual variability and functionality. A group of highly (n = 2,194) expressed RPE genes showed an overrepresentation of genes of the oxidative phosphorylation, ATP synthesis and ribosome pathways. In the group of moderately expressed genes (n = 8,776) genes of the phosphatidylinositol signaling system and aminosugars metabolism were overrepresented. As expected, the top 10 percent (n = 2,194) of genes with the highest interindividual differences in expression showed functional overrepresentation of the complement cascade, essential in inflammation in age-related macular degeneration, and other signaling pathways. Surprisingly, this same category also includes the genes involved in Bruch's membrane (BM) composition. Among the top 10 percent of genes with low interindividual differences, there was an overrepresentation of genes involved in local glycosaminoglycan turnover. Conclusion Our study expands current knowledge of the RPE transcriptome by assigning new genes, and adding data about expression level and interindividual variation. Functional annotation suggests that the RPE has high levels of protein synthesis, strong energy demands, and is exposed to high levels of oxidative stress and a variable degree of inflammation. Our data sheds new light on the molecular composition of BM, adjacent to the RPE, and is useful for

  2. The core planar cell polarity gene, Vangl2, directs adult corneal epithelial cell alignment and migration.

    PubMed

    Findlay, Amy S; Panzica, D Alessio; Walczysko, Petr; Holt, Amy B; Henderson, Deborah J; West, John D; Rajnicek, Ann M; Collinson, J Martin

    2016-10-01

    This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro. Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration.

  3. The core planar cell polarity gene, Vangl2, directs adult corneal epithelial cell alignment and migration

    PubMed Central

    Findlay, Amy S.; Panzica, D. Alessio; Walczysko, Petr; Holt, Amy B.; Henderson, Deborah J.; West, John D.; Rajnicek, Ann M.

    2016-01-01

    This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro. Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration. PMID:27853583

  4. ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

    PubMed Central

    Pipparelli, Aurélien; Arsenijevic, Yvan; Thuret, Gilles; Gain, Philippe

    2013-01-01

    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. PMID:23626771

  5. Generation of Human Female Reproductive Tract Epithelium from Human Embryonic Stem Cells

    PubMed Central

    Ye, Louie; Mayberry, Robyn; Lo, Camden Y.; Britt, Kara L.; Stanley, Edouard G.; Elefanty, Andrew G.; Gargett, Caroline E.

    2011-01-01

    Background Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT). Methodology/Principal Findings We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1+ mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA). Conclusions/Significance These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT. PMID:21698266

  6. The effects of toxicological agents on the optics and mitochondria of the lens and the mitochondria of the corneal epithelium.

    PubMed

    Bantseev, V; McCanna, D J; Driot, J-Y; Sivak, J G

    2008-04-01

    This review describes how the morphology and distribution of the mitochondria of the epithelium and the superficial fibre layers of the lens were studied using confocal scanning laser microscopy. This research was correlated with an effort to use the optical properties of the intact lens in culture as a proxy for the cornea in measuring ocular toxicity. In turn, this work led to the confocal study of the in vitro and then the in vivo cornea and their possible use in using confocal microscopy to evaluate the effect of various treatments on the integrity of the surface of the eye. Finally, confocal examination of the mitochondria of the lens has provided an avenue to the study of mitochondrial dynamics.

  7. High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

    PubMed Central

    Chng, Zhenzhi; Peh, Gary S. L.; Herath, Wishva B.; Cheng, Terence Y. D.; Ang, Heng-Pei; Toh, Kah-Peng; Robson, Paul; Mehta, Jodhbir S.; Colman, Alan

    2013-01-01

    Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type. PMID:23844023

  8. Human Bone Derived Collagen for the Development of an Artificial Corneal Endothelial Graft. In Vivo Results in a Rabbit Model

    PubMed Central

    Vázquez, Natalia; Chacón, Manuel; Rodríguez-Barrientos, Carlos A.; Merayo-Lloves, Jesús; Naveiras, Miguel; Baamonde, Begoña; Alfonso, Jose F.; Zambrano-Andazol, Iriana; Riestra, Ana C.; Meana, Álvaro

    2016-01-01

    Corneal keratoplasty (penetrating or lamellar) using cadaveric human tissue, is nowadays the main treatment for corneal endotelial dysfunctions. However, there is a worldwide shortage of donor corneas available for transplantation and about 53% of the world’s population have no access to corneal transplantation. Generating a complete cornea by tissue engineering is still a tough goal, but an endothelial lamellar graft might be an easier task. In this study, we developed a tissue engineered corneal endothelium by culturing human corneal endothelial cells on a human purified type I collagen membrane. Human corneal endothelial cells were cultured from corneal rims after corneal penetrating keratoplasty and type I collagen was isolated from remnant cancellous bone chips. Isolated type I collagen was analyzed by western blot, liquid chromatography -mass spectrometry and quantified using the exponentially modified protein abundance index. Later on, collagen solution was casted at room temperature obtaining an optically transparent and mechanically manageable membrane that supports the growth of human and rabbit corneal endothelial cells which expressed characteristic markers of corneal endothelium: zonula ocluddens-1 and Na+/K+ ATPase. To evaluate the therapeutic efficiency of our artificial endothelial grafts, human purified type I collagen membranes cultured with rabbit corneal endothelial cells were transplanted in New Zealand white rabbits that were kept under a minimal immunosuppression regimen. Transplanted corneas maintained transparency for as long as 6 weeks without obvious edema or immune rejection and maintaining the same endothelial markers that in a healthy cornea. In conclusion, it is possible to develop an artificial human corneal endothelial graft using remnant tissues that are not employed in transplant procedures. This artificial endothelial graft can restore the integrality of corneal endothelium in an experimental model of endothelial dysfunction

  9. Increased expression of nestin in human pterygial epithelium

    PubMed Central

    Wen, Dan; Wang, Hua; Heng, Boon Chin; Liu, Hua

    2013-01-01

    AIM To investigate the distribution of nestin-positive cells in pterygium, as well as the relationship between nestin-positive cells and proliferative cells in the pathogenesis of pterygium. METHODS Nine pterygium specimens and 5 normal conjunctiva specimens were investigated. All explanted specimens were immediately immersed in 5-Ethynyl-2′-deoxyuridine, and were subjected to hematoxylin and eosin staining, as well as immunostaining to detect nestin. RESULTS Small sub-populations of nestin-expressing cells in both normal and pterygial conjunctiva epithelium were found. These were located at the superficial layer of the epithelium, and were significantly increased (P=0.007) and spread out in the pterygial conjunctiva epithelium, even though these cells were mitotically quiescent. CONCLUSION In pterygium, more nestin-positive cells were present at the superficial layer of the epithelium. With growing scientific evidence that nestin plays an important role in defining various specialized cell types, such as stem cells, cancer cells and angiogenic cells, further investigations on the roles of nestin-expressing cells in pterygium may help to uncover the mechanisms of initiation, development and the prognosis of this disease. PMID:23826515

  10. Less Invasive Corneal Transepithelial Electrical Resistance Measurement Method.

    PubMed

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

    2016-01-01

    To evaluate acute corneal permeability changes after instillation of benzalkonium chloride (BAC) using a newly developed in vivo less invasive corneal transepithelial electrical resistance (TER) measurement method in animals and humans. We previously developed an in vivo method for measuring corneal TER using intraocular electrodes in animals. This method can be used to precisely measure the decline of the corneal barrier function after instillation of BAC. To lessen the invasiveness of that procedure, we further refined the method for measuring the corneal TER by developing electrodes that could be placed on the surface of the cornea and in the conjunctival sac instead of inserting them into the anterior chamber. Corneal TER changes before and after exposure to 0.02% BAC were determined in this study using the new device in both rabbits and humans. There was a significant decrease in the corneal TER after exposure of the cornea to 0.02% BAC solution in both rabbits and humans (P<.01). The results of this new less invasive method agreed with those of formerly established anterior chamber methods in rabbit experiments. This new less invasive corneal TER measurement method enables us for the first time to measure TER of the human cornea, allowing safe and reliable investigation of the direct effect of different eye drop treatments on the corneal epithelium. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Cultured corneal epithelia for ocular surface disease.

    PubMed Central

    Schwab, I R

    1999-01-01

    PURPOSE: To evaluate the potential efficacy for autologous and allogeneic expanded corneal epithelial cell transplants derived from harvested limbal corneal epithelial stem cells cultured in vitro for the management of ocular surface disease. METHODS: Human Subjects. Of the 19 human subjects included, 18 (20 procedures) underwent in vitro cultured corneal epithelial cell transplants using various carriers for the epithelial cells to determine the most efficacious approach. Sixteen patients (18 procedures on 17 eyes) received autologous transplants, and 2 patients (1 procedure each) received allogeneic sibling grafts. The presumed corneal epithelial stem cells from 1 patient did not grow in vitro. The carriers for the expanded corneal epithelial cells included corneal stroma, type 1 collagen (Vitrogen), soft contact lenses, collagen shields, and amniotic membrane for the autologous grafts and only amniotic membrane for the allogeneic sibling grafts. Histologic confirmation was reviewed on selected donor grafts. Amniotic membrane as carrier. Further studies were made to determine whether amniotic membrane might be the best carrier for the expanding corneal epithelial cells. Seventeen different combinations of tryspinization, sonication, scraping, and washing were studied to find the simplest, most effective method for removing the amniotic epithelium while still preserving the histologic appearance of the basement membrane of the amnion. Presumed corneal epithelial stem cells were harvested and expanded in vitro and applied to the amniotic membrane to create a composite graft. Thus, the composite graft consisted of the amniotic membrane from which the original epithelium had been removed without significant histologic damage to the basement membrane, and the expanded corneal epithelial stem cells, which had been applied to and had successfully adhered to the denuded amniotic membrane. Animal model. Twelve rabbits had the ocular surface of 1 eye damaged in a standard

  12. The Effect of Silica Nanoparticles on Human Corneal Epithelial Cells

    PubMed Central

    Park, Joo-Hee; Jeong, Hyejoong; Hong, Jinkee; Chang, Minwook; Kim, Martha; Chuck, Roy S.; Lee, Jimmy K.; Park, Choul-Yong

    2016-01-01

    Ocular drug delivery is an interesting field in current research. Silica nanoparticles (SiNPs) are promising drug carriers for ophthalmic drug delivery. However, little is known about the toxicity of SiNPs on ocular surface cells such as human corneal epithelial cells (HCECs). In this study, we evaluated the cytotoxicity induced by 50, 100 and 150 nm sizes of SiNPs on cultured HCECs for up to 48 hours. SiNPs were up-taken by HCECs inside cytoplasmic vacuoles. Cellular reactive oxygen species generation was mildly elevated, dose dependently, with SiNPs, but no significant decrease of cellular viability was observed up to concentrations of 100 μg/ml for three different sized SiNPs. Western blot assays revealed that both cellular autophagy and mammalian target of rapamycin (mTOR) pathways were activated with the addition of SiNPs. Our findings suggested that 50, 100 and 150 nm sized SiNPs did not induce significant cytotoxicity in cultured HCECs. PMID:27876873

  13. Scanning acoustic microscopy for mapping the microelastic properties of human corneal tissue.

    PubMed

    Beshtawi, Ithar M; Akhtar, Riaz; Hillarby, M Chantal; O'Donnell, Clare; Zhao, Xuegen; Brahma, Arun; Carley, Fiona; Derby, Brian; Radhakrishnan, Hema

    2013-04-01

    To assess the feasibility of applying scanning acoustic microscopy (SAM) on UV cross-linked corneal tissue for mapping and analyzing its biomechanical properties. Five corneal pairs (10 corneas) were used. In each pair, one cornea was cross-linked (epithelium removed, riboflavin application for 45 min and UVA irradiation for 30 min) and the contralateral control cornea was epithelial debrided and treated only with riboflavin for 45 min. Histological sections were prepared and their mechanical properties were examined using SAM. A line profile technique and 2D analysis was used to analyze the mechanical properties of the corneas. Then the corneal paraformaldehyde and unfixed sections were examined histologically using hematoxylin and eosin (H&E) staining. In the frozen fresh corneal tissue, the speed of sound of the treated corneas was 1672.5 ± 36.9 ms(-1), while it was 1584.2 ± 25.9 ms(-1) in the untreated corneas. In the paraformaldehyde fixed corneal tissue, the speed of sound of the treated corneas was 1863.0 ± 12.7 ms(-1), while it was 1739.5 ± 30.4 ms(-1) in the untreated corneas. The images obtained from the SAM technique corresponded well with the histological images obtained with H&E staining. SAM is a novel tool for examining corneal tissue with a high spatial resolution, providing both histological and mechanical data.

  14. HUMAN CORNEAL ENDOTHELIAL CELL TRANSPLANTATION IN A HUMAN EX VIVO MODEL

    PubMed Central

    Patel, Sanjay V.; Bachman, Lori A.; Hann, Cheryl R.; Bahler, Cindy K.; Fautsch, Michael P.

    2009-01-01

    Purpose To determine the effects of incorporating superparamagnetic microspheres (SPMs) into cultured human corneal endothelial cells (HCECs), and to describe preliminary experiments of HCEC transplantation, facilitated by SPMs and an external magnetic field, in a human anterior segment ex vivo model. Methods HCECs were cultured in monolayer and incorporated with magnetite oxide SPMs (900 nm, 300 nm, and 100 nm) at different concentrations. Cell viability, migration toward a magnetic field, and light transmittance were measured after incorporation of SPMs. HCEC transplantation to human recipients was investigated with anterior segments in organ culture subjected to an external magnetic field. Light and electron microscopy were used to assess HCEC attachment to corneal stroma. Results SPMs were incorporated into the cytoplasm of HCECs after overnight incubation. None of the SPMs affected the short-term viability of cultured HCECs (P>0.14, n=6) or their light transmittance (P>0.06, n=5), although there was a trend toward decreased transmittance with higher concentrations of the 900 nm SPM. Cell migration toward a magnetic field was higher for HCECs with incorporated SPMs than for HCECs without SPMs (P≤ 0.01, n=6), with dose-response relationships evident for the 300 nm and 100 nm SPMs. SPMs facilitated the attachment of HCECs to corneal stroma in the human anterior segment model with minimal change in intracameral (intraocular) pressure. Conclusions SPMs facilitate migration of HCECs toward a magnetic source and attachment of cells to corneal stroma without affecting cell viability or light transmittance. The human anterior segment model can be used to study HCEC transplantation. PMID:19136716

  15. Using optical coherence tomography to assess the role of age and region in corneal epithelium and palisades of vogt

    PubMed Central

    Lin, Hsuan-Chieh; Tew, Teck Boon; Hsieh, Yi-Ting; Lin, Szu-Yuan; Chang, Huai-Wen; Hu, Fung-Rong; Chen, Wei-Li

    2016-01-01

    Abstract Using spectral-domain optical coherence tomography (OCT) to observe the morphology and epithelial thickness (ET) of the palisades of Vogt (POV), and to evaluate the role of age and region on these structures. One hundred twelve eyes of 112 healthy subjects were enrolled and divided into 4 groups: A (0–19), B (20–39), C (40–59), and D (≥60 years old). RTvue-100 OCT was applied on the cornea and the limbus. The morphology of the subepithelial stroma underneath the epithelium of POV was classified into typical and atypical types. Maximum ET of POV was measured manually from OCT images. The positive rate of typical POV in superior, nasal, temporal, and inferior limbus was: Group A: 100%, 69.2%, 65.4%, 100%; Group B: 100%, 73.5%, 61.8%, 94.1%; Group C: 95.8%, 41.7%, 37.5%, 83.3%; Group D: 67.9%, 0%, 3.6%, 25%, showing a significant decreasing tendency with age. The maximum ET of POV in superior, nasal, temporal, and inferior limbus was: Group A: 103.5 ± 10.1 um, 89.2 ± 9.7 um, 87.9 ± 13.6 um, 104.7 ± 14.1 um; Group B: 111.4 ± 15.8 um, 85.3 ± 9.9 um, 88.2 ± 8.6 um, 112.6 ± 19.7 um; Group C: 116.4 ± 16.4 um, 82.8 ± 11.6 um, 87.0 ± 11.6 um, 120.0 ± 25.6 um; Group D: 96.3 ± 17.9 um, 73.8 ± 15.9 um, 79.2 ± 16.7 um, 87.4 ± 18.5 um. Age-dependent change was observed. In general, the maximum ET of POV in superior/inferior quadrants was thicker than the other 2 quadrants. Spectral-domain OCT is a useful tool to observe the limbal microstructure and provide invaluable information. Aging and anatomic regions had significant effects on the microstructure of these areas. PMID:27583846

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

    PubMed

    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.

  17. Corneal Equilibrium Flux as a Function of Corneal Surface Oxygen Tension.

    PubMed

    Compañ, Vicente; Aguilella-Arzo, Marcel; Weissman, Barry A

    2017-06-01

    Oxygen is essential for aerobic mammalian cell physiology. Oxygen tension (PO2) should reach a minimum at some position within the corneal stroma, and oxygen flux should be zero, by definition, at this point as well. We found the locations and magnitudes of this "corneal equilibrium flux" (xmin) and explored its physiological implications. We used an application of the Monod kinetic model to calculate xmin for normal human cornea as anterior surface PO2 changes from 155 to 20 mmHg. We find that xmin deepens, broadens, and advances from 1.25 μm above the endothelial-aqueous humor surface toward the epithelium (reaching a position 320 μm above the endothelial-aqueous humor surface) as anterior corneal surface PO2 decreases from 155 to 20 mmHg. Our model supports an anterior corneal oxygen flux of 9 μL O2 · cm · h and an epithelial oxygen consumption of approximately 4 μL O2 · cm · h. Only at the highest anterior corneal PO2 does our model predict that oxygen diffuses all the way through the cornea to perhaps reach the anterior chamber. Of most interest, corneal oxygen consumption should be supported down to a corneal surface PO2 of 60 to 80 mmHg but declines below this range. We conclude that the critical oxygen tension for hypoxia induced corneal swelling is more likely this range rather than a fixed value.

  18. Corneal Ring Infiltrates Caused by Serratia marcescens in a Patient with Human Immunodeficiency Virus

    PubMed Central

    Chaidaroon, Winai; Supalaset, Sumet

    2016-01-01

    Purpose To describe corneal ring infiltrates caused by Serratia marcescens in a patient with human immunodeficiency virus (HIV-1) who wore contact lenses. Methods A case study of a patient with keratitis due to an infection caused by S. marcescens and exhibiting corneal ring infiltrates was reviewed for history, clinical manifestation, microscopic study, and management. Results A 29-year-old man who had a history of contact lens wear and HIV-1 infection was admitted to hospital because of blurred vision, redness, and corneal infiltrates in the shape of a ring in the left eye. The visual acuity (VA) in both eyes was hand movement (uncorrected). Corneal scrapings were performed. The culture results of the corneal specimens revealed S. marcescens. The culture results of the contact lens disclosed the same organism. The corneal ulcer responded well to treatment with topical gentamycin sulfate 14 mg/ml. The final VA remained hand movement. Conclusions S. marcescens can cause ring infiltrates in a HIV-1 patient who wears contact lenses. The treatment result for S. marcescens keratitis in a HIV-1 patient who wore contact lenses was favorable after intensive use of fortified topical antibiotics. PMID:27721784

  19. Vascular endothelial growth factor receptor-1 (VEGFR-1) expression in human corneal fibroblast decreased with age

    PubMed Central

    Berthaut, Alexandre; Mirshahi, Pezhman; Benabbou, Nadia; Azzazene, Dalel; Bordu, Camille; Therwath, Amu; Legeais, Jean-marc

    2009-01-01

    Purpose Mechanisms by which fibroblast networks between stromal lamellae are laid in the corneal stroma are far from clear. We have investigated the role of vascular endothelial growth factor receptors (VEGFRs) by in vitro studies in the human corneal network formation obtained from donors whose ages ranged from 19 to 89 years. Methods Corneal fibroblasts were prepared from cornea donations. The functional properties of these cells to form networks were analyzed using a semi solid matrix (substratum) of Matrigel™. The presence of VEGF receptor-1 (VEGFR-1) and the functionality in these fibroblasts were investigated using immunofluorescence, molecular analysis (gene microarray, reverse transcription polymerase chain reaction [RT–PCR] and VEGFR siRNA transfections), and cell culture. Results Corneal fibroblasts from 61 donors were classified into two groups according to whether they formed (82%) a reticulum on Matrigel™ or not (18%). By RT–PCR and immunofluorescence analysis, we showed that corneal fibroblasts expressed VEGFR-1 (mRNA and protein). Further, cell culture analysis revealed that only the network (reticulum) forming corneal fibroblast expressed VEGFR-1 in contrast to non network-forming fibroblasts. Use of inhibitors such as VEGFR-1 siRNA transfection or neutralizing antibody (Avastin™) indicated that VEGFR-1 was essential to the formation of the corneal network in vitro. Conclusions The cell reticulum formation seemed to be directly related to the expression of VEGFR-1 in the corneal fibroblast, and this expression decreased with age. The decrease in VEGFR-1 expression is probably related to the diminution of autocrine functions, which may alter the overall tissular homeostasis. This may culminate in the gradual development of poor vision, which is observed in certain pathologies and in aging individuals. PMID:19816604

  20. Identification of distinct layers within the stratified squamous epithelium of the adult human true vocal fold.

    PubMed

    Dowdall, Jayme R; Sadow, Peter M; Hartnick, Christopher; Vinarsky, Vladimir; Mou, Hongmei; Zhao, Rui; Song, Phillip C; Franco, Ramon A; Rajagopal, Jayaraj

    2015-09-01

    A precise molecular schema for classifying the different cell types of the normal human vocal fold epithelium is lacking. We hypothesize that the true vocal fold epithelium has a cellular architecture and organization similar to that of other stratified squamous epithelia including the skin, cornea, oral mucosa, and esophagus. In analogy to disorders of the skin and gastrointestinal tract, a molecular definition of the normal cell types within the human vocal fold epithelium and a description of their geometric relationships should serve as a foundation for characterizing cellular changes associated with metaplasia, dysplasia, and cancer. Qualitative study with adult human larynges. Histologic sections of normal human laryngeal tissue were analyzed for morphology (hematoxylin and eosin) and immunohistochemical protein expression profile, including cytokeratins (CK13 and CK14), cornified envelope proteins (involucrin), basal cells (NGFR/p75), and proliferation markers (Ki67). We demonstrated that three distinct cell strata with unique marker profiles are present within the stratified squamous epithelium of the true vocal fold. We used these definitions to establish that cell proliferation is restricted to certain cell types and layers within the epithelium. These distinct cell types are reproducible across five normal adult larynges. We have established that three layers of cells are present within the normal adult stratified squamous epithelium of the true vocal fold. Furthermore, replicating cell populations are largely restricted to the parabasal strata within the epithelium. This delineation of distinct cell populations will facilitate future studies of vocal fold regeneration and cancer. N/A. © 2015 The American Laryngological, Rhinological and Otological Society, Inc.

  1. Rapid, automated mosaicking of the human corneal subbasal nerve plexus.

    PubMed

    Vaishnav, Yash J; Rucker, Stuart A; Saharia, Keshav; McNamara, Nancy A

    2017-03-04

    Corneal confocal microscopy (CCM) is an in vivo technique used to study corneal nerve morphology. The largest proportion of nerves innervating the cornea lie within the subbasal nerve plexus, where their morphology is altered by refractive surgery, diabetes and dry eye. The main limitations to clinical use of CCM as a diagnostic tool are the small field of view of CCM images and the lengthy time needed to quantify nerves in collected images. Here, we present a novel, rapid, fully automated technique to mosaic individual CCM images into wide-field maps of corneal nerves. We implemented an OpenCV image stitcher that accounts for corneal deformation and uses feature detection to stitch CCM images into a montage. The method takes 3-5 min to process and stitch 40-100 frames on an Amazon EC2 Micro instance. The speed, automation and ease of use conferred by this technique is the first step toward point of care evaluation of wide-field subbasal plexus (SBP) maps in a clinical setting.

  2. The 3895-bp mitochondrial DNA deletion in the human eye: a potential involvement in corneal ageing and macular degeneration.

    PubMed

    Gendron, Sébastien P; Bastien, Nathalie; Mallet, Justin D; Rochette, Patrick J

    2013-03-01

    In human skin, the 3895-bp deletion of mitochondrial DNA (mtDNA(3895)) is catalysed by ultraviolet (UV) light through the generation of reactive oxygen species. Given its function in vision, the human eye is exposed to oxidising UV and blue light in its anterior (cornea, iris) and posterior (retina) structures. In this study, we employed a highly sensitive quantitative PCR technique to determine mtDNA(3895) occurrence in human eye. Our analysis shows that the mtDNA(3895) is concentrated in both the cornea and the retina. Within the cornea, the highest mtDNA(3895) level is found in the stroma, the cellular layer conferring transparency and rigidity to the human cornea. Moreover, mtDNA(3895) accumulates with age in the stroma, suggesting a role of this deletion in corneal ageing. Within the retina, mtDNA(3895) is concentrated in the macular region of both the neural retina and the retinal pigment epithelium, supporting the hypothesis that this deletion is implicated in retinal pathologies such as age-related macular degenerescence. Taken together, our results imply that UV and blue light catalyse mtDNA(3895) induction in the human eye.

  3. In situ immunohistochemical study of Bcl-2 and heat shock proteins in human corneal endothelial cells during corneal storage

    PubMed Central

    Gain, P.; Thuret, G.; Chiquet, C.; Dumollard, J. M.; Mosnier, J. F.; Campos, L.

    2001-01-01

    AIM—To investigate the expression of Bcl-2 and heat shock proteins (HSPs), which are known to increase cell survival, in human corneal endothelial cells (HCECs) of corneas stored in organ culture.
METHODS—32 paired corneas were randomly assigned to either a short or a long storage time. The flat mounts of endothelium were examined after immunostaining with monoclonal antibodies to Bcl-2 and HSP 27, 60, 70, and 90.
RESULTS—HCECs expressed generally all the proteins studied. Bcl-2 expression was weaker in the long stored corneas (p=0.035). There was no relation between immunostaining, age, sex, or death to culture time. Frequently some Descemet membranes carried negative cells preferentially located in folds and exhibiting morphological changes consistent with swelling cells corresponding to early stages of apoptosis.
CONCLUSION—Expression of these cytoprotective proteins reflects the high level of HCEC resistance to stresses induced by organ culture. The decreased immunostaining of Bcl-2 in the long storage group could act in cellular loss currently observed with storage time. The negativity of Bcl-2 and HSP labelling in corneal folding may be related to apoptosis.

 PMID:11466261

  4. Influence of sex on gene expression in human corneal epithelial cells

    PubMed Central

    Suzuki, Tomo; Richards, Stephen M.; Liu, Shaohui; Jensen, Roderick V.

    2009-01-01

    Purpose Sex-associated differences have been identified in the anatomy, physiology and pathophysiology of the human cornea. We hypothesize that many of these differences are due to fundamental variations in gene expression. Our objective in this study was to determine whether such differences exist in human corneal epithelial cells both in vivo and in vitro. Methods Human corneal epithelial cells were isolated from the corneoscleral rims of male and female donors. Cells were processed either directly for RNA extraction, or first cultured in phenol red-free keratinocyte serum-free media. The RNA samples were examined for differentially expressed mRNAs by using of CodeLink Bioarrays and Affymetrix GeneChips. Data were analyzed with GeneSifter.Net software. Results Our results demonstrate that sex significantly influences the expression of over 600 genes in human corneal epithelial cells in vivo. These genes are involved in a broad spectrum of biologic processes, molecular functions and cellular components, such as metabolic processes, DNA replication, cell migration, RNA binding, oxidoreductase activity and nucleoli. We also identified significant, sex-related effects on gene expression in human corneal epithelial cells in vitro. However, with few exceptions (e.g., X- and Y-linked genes), these sex-related differences in gene expression in vitro were typically different than those in vivo. Conclusions Our findings support our hypothesis that sex-related differences exist in the gene expression of human corneal epithelial cells. Variations in gene expression may contribute to sex-related differences in the prevalence of certain corneal diseases. PMID:20011627

  5. Effects of formaldehyde on xenotransplanted human respiratory epithelium. Research report, August 1986-April 1991

    SciTech Connect

    Klein-Szanto, A.J.P.; Ura, H.; Momiki, S.; Bonfil, D.; Litwin, S.

    1992-07-01

    A laboratory animal model that permits the exposure of xenotransplanted human respiratory epithelium to formaldehyde was used to study the effects of formaldehyde alone or in combination with a carcinogenic metabolite of benzo(a)pyrene, benzo(a)pyrene diol epoxide. Epithelial cells obtained from autopsies of 20 full-term human fetuses or infants less than one year old were isolated, amplified in vitro, inoculated into rat tracheas from which the epithelial layer had been removed, and then transplanted subcutaneously into irradiated athymic nude mice. Four weeks after transplantation, the tracheal transplants were implanted with silastic devices containing 0, 0.5, 1, or 2 mg of powdered formaldehyde (Study 1). The tracheal transplants were examined histologically 2, 4, 8, or 16 weeks after transplantation. Before killing, all animals were injected with a single pulse of tritiated thymidine. A maximum effect was visible two weeks after exposure; 2 mg of formaldehyde produced numerous areas of epithelial erosion and inflammation. All doses produced areas of hyperplastic epithelium and areas of atrophic epithelium. The labeling indices showed dose dependence between two and four weeks after the initiation of exposure. These studies show that low doses of formaldehyde can elicit a proliferative response in human infant tracheobronchial epithelium that is not preceded by a massive toxic effect. Similar studies using xenotransplanted human adult nasal respiratory epithelium (Study 2) showed a response pattern similar to that of Study 1. In Study 3, using cells from 11 human infants, formaldehyde applied simultaneously or sequentially with benzo(a)pyrene diol epoxide did not induce epithelial alterations different from those observed with formaldehyde alone. This indicated that, under the doses and exposure conditions used, it was not possible to observe any cocarcinogenic or promoting effects of formaldehyde on the human infant tracheobronchial epithelium.

  6. Isolation and chromosomal localization of a cornea-specific human keratin 12 gene and detection of four mutations in Meesmann corneal epithelial dystrophy.

    PubMed Central

    Nishida, K; Honma, Y; Dota, A; Kawasaki, S; Adachi, W; Nakamura, T; Quantock, A J; Hosotani, H; Yamamoto, S; Okada, M; Shimomura, Y; Kinoshita, S

    1997-01-01

    Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I keratin. Images Figure 1 Figure 2 Figure 3 PMID:9399908

  7. Light scattering from human corneal grafts: Bulk and surface contribution

    NASA Astrophysics Data System (ADS)

    Latour, Gaël; Georges, Gaëlle; Lamoine, Laure Siozade; Deumié, Carole; Conrath, John; Hoffart, Louis

    2010-09-01

    The cornea is the only transparent tissue in the body. The transparency is the main characteristic of the corneal tissue, and depends not only on the transmission coefficient but also on the losses by scattering and absorption. The scattering properties of the cornea tissues become one of the most important parameters in the case of the corneal graft. These scattering properties are studied in this paper in the reflected half area, similar to the diagnosis configuration. We quantify the influence of the cornea thickness and of the epithelial layer on scattering level. The technique of ellipsometry on scattered field is also used to analyze the polarization properties in order to determine the origin of scattering (surface and/or bulk).

  8. Isolated corneal papilloma-like lesion associated with human papilloma virus type 6.

    PubMed

    Park, Choul Yong; Kim, Eo-Jin; Choi, Jong Sun; Chuck, Roy S

    2011-05-01

    To report a case of a corneal papilloma-like lesion associated with human papilloma virus type 6. A 48-year-old woman presented with a 2-year history of ocular discomfort and gradual visual deterioration in her right eye. Ophthalmic examination revealed an elevated, semitranslucent, well-defined vascularized mass approximately 4 × 2.5 mm in size localized to the right cornea. The surface of the mass appeared smooth and many small, shallow, and irregular elevations were noted. An excisional biopsy was performed. The underlying cornea was markedly thinned, and fine ramifying vasculature was also noted on the exposed corneal stroma. Typical koilocytic change was observed on the histopathologic examination. Polymerase chain reaction revealed the existence of human papilloma virus type 6 DNA. Here we describe a case of an isolated corneal papilloma-like lesion. Although the corneal extension of the limbal or the conjunctival papillomas has been commonly observed, an isolated corneal papilloma-like lesion with underlying stromal destruction has only rarely been reported.

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

    PubMed

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

    2008-02-01

    The purpose of this study was to evaluate the potential value of different epithelial cell culture systems as in vitro models for studying corneal permeability. Transformed human corneal epithelial (HCE-T) cells and Statens Serum Institut rabbit corneal (SIRC) cells were cultured on permeable filters. SkinEthic human corneal epithelium (S-HCE) and Clonetics human corneal epithelium (C-HCE) were received as ready-to-use systems. Excised rabbit corneas (ERCs) and human corneas (EHCs) were mounted in Ussing chambers, and used as references. Barrier properties were assessed by measuring transepithelial electrical resistance, and by determining the apparent permeability of markers with different physico-chemical properties, namely, fluorescein, sodium salt; propranolol hydrochloride; moxaverine hydrochloride; timolol hydrogenmaleate; and rhodamine 123. SIRC cells and the S-HCE failed to develop epithelial barrier properties, and hence were unable to distinguish between the permeation markers. Barrier function and the power to differentiate compound permeabilities were evident with HCE-T cells, and were even more pronounced in the case of C-HCE, corresponding very well with data from ERCs and EHCs. A net secretion of rhodamine 123 was not observed with any of the models, suggesting that P-glycoprotein or similar efflux systems have no significant effects on corneal permeability. Currently available corneal epithelial cell culture systems show differences in epithelial barrier function. Systems lacking functional cell-cell contacts are of limited value for assessing corneal permeability, and should be critically evaluated for other purposes.

  10. Glycoconjugate secretion in human airways in vitro: effects of epithelium removal.

    PubMed Central

    Sossé-Alaoui, H; Labat, C; Gorenne, I; Thomas de Montpreville, V; Bara, J; Brink, C

    1998-01-01

    The aim of this study was to examine glycoconjugate secretion in human airways with and without an epithelium. Glycoconjugate release in supernatants derived from human airways in vitro was determined using an ELISA assay with an anti-human mucin monoclonal antibody (MAb 3D3). This monoclonal antibody reacted strongly with Le(b) antigen but also recognized in vitro Le(a) and Le(y) determinants. In 11 of the 34 different lung samples (32%) studied the glycoconjugate levels were below the threshhold of detection for this assay. The mean basal secretion of glycoconjugates in human airways in vitro was 100+/-28 microg/g tissue (Period I; n = 23 different lung samples). The amount of glycoconjugate measured in the medium derived from human isolated bronchial ring preparations did not change under control conditions during the course of the experimental procedure (Period I; 128+/-46 microg/g tissue and Period II; 159 +/-48 microg/g tissue; n = 13 paired lung samples). In the supernatants of airway preparations with an intact epithelium the amount of glycoconjugates detected was 90+/-38 microg/g tissue (Period I; n = 12 different lung samples) and removal of the epithelium did not alter this basal glycoconjugate release (94+/-60 microg/g tissue: Period I, n = 8 different lung samples). The absence of the epithelial layer was confirmed by histological evaluation. Methacholine (100 microM) induced a 10- and four-fold increase in glycoconjugate release from airways with and without an epithelium, respectively. In contrast, in preparations with an epithelium, LTD4 (10 microM) and anti-IgE (dilution: 1/1000) did not cause an increase of glycoconjugate release. The methacholine difference between airways with and without an epithelium was not significantly different (P > 0.10). However, a treatment with atropine (100 microM) prevented the increase of glycoconjugate release in preparations with an epithelium. These data derived from a limited number of experiments suggest that

  11. The expression of human corneal MMP-2, MMP-9, proMMP-13 and TIMP-1 in bullous keratopathy and keratoconus.

    PubMed

    Predović, Jurica; Balog, Tihomir; Marotti, Tanja; Gabrić, Nikica; Bohac, Maja; Romac, Ivana; Dekaris, Iva

    2008-10-01

    We aim to find a link between keratokonus (KC) and bullous keratopathy (BK), and extra cellular matrix re-modellation molecules. The activities of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), pro-matrix metalloproteinase-13 (proMMP-13) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) were measured using immunoassay in three human corneal tissue layers (epithelium, stroma and endothelium) supernatants of the patients with KC and BK which underwent the perforative keratoplasty. MPP-2, MMP-9, proMMP-13 and TIMP-1 activity was detected in all samples. The epithelial layer showed significantly higher levels of MMP-9 and proMMP-13 in BK than in KC. Increased levels of MMP-2 (p=0.07) levels were found in bullous keratopathy compared to keratoconus patients. Epithelial TIMP-1 showed no significant difference in activity between KC and BK. All these findings suggest an active degradation of the extra-cellular matrix in epithelial corneal layer in Bullous Keratopathy. No difference in the concentration of MMP-2, MMP-9, proMMP-13 and TIMP-1 between KC and BK in corneal stroma and endothelium suggest that neither of these molecules play important role in KC or BK pathogenesis, at least not in stroma and endothelium.

  12. Analysis of human cytomegalovirus replication in primary cultured human corneal endothelial cells

    PubMed Central

    Hosogai, Mayumi; Shima, Nobuyuki; Nakatani, Yoko; Inoue, Teruki; Iso, Tatsuya; Yokoo, Hideaki; Yorifuji, Hiroshi; Akiyama, Hideo; Kishi, Shoji; Isomura, Hiroki

    2015-01-01

    Background/aims Since the first case of human cytomegalovirus (HCMV)-induced corneal endotheliitis in which HCMV DNA was detected from the patient's aqueous humour using PCR, the clinical evidence for HCMV endotheliitis has been accumulating. However, it remains to be confirmed whether HCMV can efficiently replicate in corneal endothelial cells. We, therefore, sought to determine whether primary cultured human corneal endothelial cells (HCECs) could support HCMV replication. Methods Human foreskin fibroblasts (HFFs) have been shown to be fully permissive for HCMV replication, and are commonly used as an in vitro model for HCMV lytic replication. Therefore, primary cultured HCECs or HFFs were infected with the vascular endotheliotropic HCMV strain TB40/E or laboratory strain Towne. We then compared viral mRNA and protein expression, genome replication and growth between the TB40/E-infected and Towne-infected HCECs and HFFs. Results When HCECs were infected with TB40/E or Towne, rounded cells resembling owl's eyes as well as viral antigens were detected. Viral mRNA synthesis and protein expression proceeded efficiently in the HCECs and HFFs infected with TB40/E or Towne at a high multiplicity of infection (MOI). Similarly, the viral genome was also effectively replicated, with UL44—a viral DNA polymerase processivity factor—foci observed in the nuclei of HCECs. HCECs produced a substantial number of infectious virions after infection with TB40/E at both a high and low MOI. Conclusions Primary cultured HCECs could efficiently support HCMV replication after infection at both a high and low MOI. PMID:26261231

  13. Transient receptor potential vanilloid 4 (TRPV4) silencing in Helicobacter pylori-infected human gastric epithelium.

    PubMed

    Mihara, Hiroshi; Suzuki, Nobuhiro; Muhammad, Jibran Sualeh; Nanjo, Sohachi; Ando, Takayuki; Fujinami, Haruka; Kajiura, Shinya; Hosokawa, Ayumu; Sugiyama, Toshiro

    2017-04-01

    Helicobacter pylori (HP) infection induces methylation silencing of specific genes in gastric epithelium. Various stimuli activate the nonselective cation channel TRPV4, which is expressed in gastric epithelium where it detects mechanical stimuli and promotes ATP release. As CpG islands in TRPV4 are methylated in HP-infected gastric epithelium, we evaluated HP infection-dependent changes in TRPV4 expression in gastric epithelium. Human gastric biopsy samples, a human gastric cancer cell line (AGS), and a normal gastric epithelial cell line (GES-1) were used to detect TRPV4 mRNA and protein expression by RT-PCR and Western blotting, respectively. Ca(2+) imaging was used to evaluate TRPV4 ion channel activity. TRPV4 methylation status was assessed by methylation-specific PCR (MSP). ATP release was measured by a luciferin-luciferase assay. TRPV4 mRNA and protein were detected in human gastric biopsy samples and in GES-1 cells. MSP and demethylation assays showed TRPV4 methylation silencing in AGS cells. HP coculture directly induced methylation silencing of TRPV4 in GES-1 cells. In human samples, HP infection was associated with TRPV4 methylation silencing that recovered after HP eradication in a time-dependent manner. HP infection-dependent DNA methylation suppressed TRPV4 expression in human gastric epithelia, suggesting that TRPV4 methylation may be involved in HP-associated dyspepsia. © 2016 The Authors. Helicobacter Published by John Wiley & Sons Ltd.

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

    PubMed

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

    2017-02-01

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

  15. Neuropeptides released from trigeminal neurons promote the stratification of human corneal epithelial cells.

    PubMed

    Ko, Ji-Ae; Mizuno, Yukari; Ohki, Chihiro; Chikama, Tai-ichiro; Sonoda, Koh-Hei; Kiuchi, Yoshiaki

    2014-01-07

    To examine the effects of neural cells on the stratification of and junctional protein expression by corneal epithelial cells with a coculture system. PC12 cells induced to undergo neuronal differentiation or rat trigeminal nerve cells were cultured together with simian virus 40-transformed human corneal epithelial (HCE) cells on opposite sides of a collagen vitrigel membrane. Stratification of HCE cells was examined by immunofluorescence analysis with antibodies to zonula occludens-1. Expression of junctional proteins in HCE cells was assessed by RT-PCR and immunoblot analyses. The presence of neural cells (PC12 cells or trigeminal neurons) markedly promoted the stratification of HCE cells as well as increased the amounts of N-cadherin mRNA and protein in these cells. These effects of the neural cells were mimicked by conditioned medium prepared from differentiating PC12 cells or by the neuropeptides substance P and calcitonin gene-related peptide (CGRP). Furthermore, the stimulatory effects of trigeminal neurons on the stratification of and N-cadherin expression by HCE cells were inhibited by antagonists of substance P or of CGRP. These results suggest that trigeminal neurons play an important role in the differentiation of corneal epithelial cells. Neuropeptides released from these neurons may thus regulate adhesion between corneal epithelial cells and thereby contribute to the establishment and maintenance of corneal structure and function.

  16. Anterior corneal and internal contributions to peripheral aberrations of human eyes

    NASA Astrophysics Data System (ADS)

    Atchison, David A.

    2004-03-01

    Anterior corneal and internal component contributions to overall peripheral aberrations of five human eyes were determined, based on corneal topography and overall aberration measurements. Anterior corneal position and orientation (tilt) were referenced to the line of sight. Ray tracing was performed through the anterior cornea for 6-mm-diameter pupils at angles out to 40° in both the temporal and the nasal visual fields. In general, both component and overall Zernike aberrations were greater for the nasal than for the temporal visual field. In general, the anterior corneal aberration components were considerably higher than the overall aberrations across the visual field and were balanced to a considerable degree by the internal ocular aberration components. The component and overall levels of Zernike third-order aberrations showed linear trends away from the fixation axis, and the component levels of Zernike fourth-order aberrations showed quadratic trends away from the fixation axis. The second-order, but not higher-order, aberration components were susceptible to the choice of image radius of curvature, while disregarding corneal position and orientation affected second- and higher-order aberration components.

  17. Effects of vocal fold epithelium removal on vibration in an excised human larynx model

    PubMed Central

    Tse, Justin R.; Zhang, Zhaoyan; Long, Jennifer L.

    2015-01-01

    This study investigated the impact of selective epithelial injury on phonation in an excised human larynx apparatus. With intact epithelium, the vocal folds exhibited a symmetrical vibration pattern with complete glottal closure during vibration. The epithelium was then enzymatically removed from one, then both vocal folds, which led to left-right asymmetric vibration and a decreased closed quotient. Although the mechanisms underlying these vibratory changes are unclear, these results demonstrate that some component of an intact surface layer may play an important role in achieving normal symmetric vibration and glottal closure. PMID:26233062

  18. Measurement of the topography of human cadaver lenses using the PAR corneal topography system

    NASA Astrophysics Data System (ADS)

    Fernandez, Viviana; Manns, Fabrice; Zipper, Stanley; Sandadi, Samith; Hamaoui, Marie; Tahi, Hassan; Ho, Arthur; Parel, Jean-Marie A.

    2001-06-01

    To measure the radius of curvature and asphericity of the anterior and posterior surfaces of crystalline lenses of human Eye-Bank eyes using the PAR Corneal Topography System. The measured values will be used in an optical model of the eye for lens refilling procedures.

  19. Robust bioengineered 3D functional human intestinal epithelium

    PubMed Central

    Chen, Ying; Lin, Yinan; Davis, Kimberly M.; Wang, Qianrui; Rnjak-Kovacina, Jelena; Li, Chunmei; Isberg, Ralph R.; Kumamoto, Carol A.; Mecsas, Joan; Kaplan, David L.

    2015-01-01

    Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments. PMID:26374193

  20. Robust bioengineered 3D functional human intestinal epithelium.

    PubMed

    Chen, Ying; Lin, Yinan; Davis, Kimberly M; Wang, Qianrui; Rnjak-Kovacina, Jelena; Li, Chunmei; Isberg, Ralph R; Kumamoto, Carol A; Mecsas, Joan; Kaplan, David L

    2015-09-16

    Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments.

  1. [Toxicity of topical ocular anti-allergic agents on human corneal epithelial cells in vitro].

    PubMed

    Li, Jun; Yan, Xiao-ming

    2010-01-01

    .1 +/- 5.1)%, respectively. The q values of A to B group and A to C group at 6 h were 3.27, 4.31, respectively (P < 0.05). The green fluorescent staining of calcein-AM indicated intracellular esterase activity was decreased after incubation with increasing concentration and longer exposure time. There was no significantly different result between each group (P > 0.05). The proportion of green staining cell of A, B and C group at 24 h were 100.0% with 50.0% concentration and were (23.2 +/- 4.6)%, (29.5 +/- 5.2)%, (31.1 +/- 5.5)% respectively with 20.0% concentration (F = 1.97, P = 0.377). (3) MTT assay: The results of the three groups revealed cell viability decreased significantly with increasing concentration and longer exposure time at all the concentrations except 0.8%. MTT values for A, B and C group at the concentration of 20.0%, at 6 h were 0.429 +/- 0.028, 0.367 +/- 0.038, 0.379 +/- 0.012 and 4% at 24 h were 0.457 +/- 0.025, 0.401 +/- 0.008, 0.387 +/- 0.012, respectively. The data for olopatadine 0.1% were significantly improved over those of ketotifen fumarate 0.025% and pemirolast potassium 0.1%. The q value of A to B group, A to C group were 3.01, 3.77 (P < 0.05) at the concentration of 20.0%, 6 h and were 3.63, 4.11 (P < 0.05) at the concentration, 24 h. There were no statistical significant results at other concentrations. The topical ocular anti-allergic agent, olopatadine 0.1%, showed less toxic effects on human corneal epithelial cells compared to ketotifen fumarate 0.025% and pemirolast potassium 0.1%. Olopatadine 0.1% may offer a safer option to the corneal epithelium when used to treat allergic keratoconjunctivitis over an extended period of time.

  2. Cytotoxicity of atropine to human corneal epithelial cells by inducing cell cycle arrest and mitochondrion-dependent apoptosis.

    PubMed

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

    2015-10-01

    Atropine is an anticholinergic drug for mydriasis in eye clinic, and its abuse might be cytotoxic to the cornea and result in blurred vision. However, the cytotoxicity of atropine to the cornea and its cellular and molecular mechanisms remain unknown. In this study, we investigated the cytotoxicity of atropine to corneal epithelium and its underlying mechanisms using an in vitro model of non-transfected human corneal epithelial (HCEP) cells. Our results showed that atropine, above the concentration of 0.3125 g/l (1/32 of its therapeutic dosage in eye clinic), had a dose- and time-dependent toxicity to HCEP cells by inducing morphological abnormality, cytopathic effect, viability decline, and proliferation retardation. Moreover, the proliferation-retarding effect of atropine on the cells was achieved by inducing G1/S phase arrest and downregulation of E-cadherin and β-catenin. Besides, atropine also had an apoptosis-inducing effect on the cells by inducing phosphatidylserine externalization, plasma membrane permeability elevation, DNA fragmentation and apoptotic body formation. Furthermore, atropine could also induce activations of caspase-2, -3 and -9, disruption of mitochondrial transmembrane potential, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor, implying a death receptor-mediated mitochondrion-dependent pathway is most probably involved in the apoptosis of HCEP cells induced by atropine. Taken together, our results suggest that atropine has remarkable cytotoxicity to HCEP cells by inducing cell cycle arrest and death receptor-mediated mitochondrion-dependent apoptosis.

  3. Rho GTPases and Regulation of Cell Migration and Polarization in Human Corneal Epithelial Cells

    PubMed Central

    Hou, Aihua; Toh, Li Xian; Gan, Kah Hui; Lee, Khee Jin Ryan; Manser, Edward; Tong, Louis

    2013-01-01

    Purpose Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. Methods Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. Results Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. Conclusion Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells. PMID:24130842

  4. Cytostatic and cytotoxic effects of 5-fluorouracil on human corneal epithelial cells and keratocytes.

    PubMed

    Midena, Edoardo; Lazzarini, Daniela; Catania, Anton Giulio; Moretto, Erika; Fregona, Iva; Parrozzani, Raffaele

    2013-03-01

    To investigate the effects of various 5-fluorouracil (5-FU) concentrations, exposure times, and application techniques on in vitro-cultured human corneal cells. Human corneal epithelial cell (HCEC) and human corneal keratocyte (HCK) cultures were exposed to different 5-FU concentrations (0.025%-1%) and incubation durations (5 minutes to 2 hours). The cytostatic effect was evaluated as the percentage of inhibition of migration relative to the control. The evaluation of cytotoxic effect included both phase contrast microscopic observations and viability measures performed using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] colorimetric assay. The results are expressed as ratio of optical density (OD) reduction 24 hours after exposure. The cytostatic effect was time and dose dependent. The 50% inhibiting dose was 0.55% after 1 hour of incubation for HCECs and was 0.5% after 2 hours of incubation for HCKs. A 100% inhibitory effect was never observed at any concentration or incubation duration. No cytotoxic changes were observed using an 5-FU concentration of <1%; 1% 5-FU showed time-dependent cytotoxic changes in HCEC cultures only. MTT analysis showed no OD reduction at 5-FU concentrations of <1%, whereas 1% 5-FU showed OD reduction <50% at any tested exposure time. HCECs showed higher reduction in OD than HCKs. 5-FU formulations topically used in clinical practice showed limited toxicity in normal cultured corneal epithelial cells and keratocytes.

  5. Mucin gene expression in human middle ear epithelium.

    PubMed

    Kerschner, Joseph Edward

    2007-09-01

    To investigate the expression of recently identified human mucin genes in human middle ear epithelial (MEE) specimens from in vivo middle ear (ME) tissue and to compare this mucin gene expression with mucin gene expression in an immortalized cell culture in vitro source of human MEE. Human MEE was harvested as in vivo specimens, and human MEE cell cultures were established for in vitro experimentation. RNA was extracted from MEE and primers designed for reverse-transcription polymerase chain reaction to assess for mucin gene MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC11, MUC12, MUC13, MUC15, MUC16, MUC18, MUC19, and MUC20 expression. Mucin gene expression in the in vivo and in vitro ME tissue was compared against tissues with known expression of the mucin genes in question. Mucin genes MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC8, MUC9, MUC11, MUC13, MUC15, MUC16, MUC18, MUC19, and MUC20 were identified and expressed in both the in vivo and in vitro samples of MEE. Mucin genes MUC6, MUC12, and MUC17 were not identified in either tissue samples. Many of the mucin genes that have been recently identified are expressed in human MEE. These genes are expressed in a similar manner in both in vivo and in vitro models. Understanding the mechanisms in which these genes regulate the physiology and pathophysiology of MEE will provide a more thorough understanding of the molecular mechanics of the MEE and disease conditions such as otitis media.

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

    PubMed

    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.

  7. Discovery of Molecular Markers to Discriminate Corneal Endothelial Cells in the Human Body

    PubMed Central

    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. PMID:25807145

  8. Candida tropicalis biofilm and human epithelium invasion is highly influenced by environmental pH.

    PubMed

    Ferreira, Carina; Gonçalves, Bruna; Vilas Boas, Diana; Oliveira, Hugo; Henriques, Mariana; Azeredo, Joana; Silva, Sónia

    2016-11-01

    The main goal of this study was to investigate the role of pH on Candida tropicalis virulence determinants, namely the ability to form biofilms and to colonize/invade reconstituted human vaginal epithelia. Biofilm formation was evaluated by enumeration of cultivable cells, total biomass quantification and structural analysis by scanning electron microscopy and confocal laser scanning microscopy. Candida tropicalis human vaginal epithelium colonization and invasiveness were examined qualitatively by epifluorescence microscopy and quantitatively by a novel quantitative real-time PCR protocol for Candida quantification in tissues. The results revealed that environmental pH influences C. tropicalis biofilm formation as well as the colonization and potential to invade human epithelium with intensification at neutral and alkaline conditions compared to acidic conditions. For the first time, we have demonstrated that C. tropicalis biofilm formation and invasion is highly influenced by environmental pH. © Crown copyright 2016.

  9. Sodium-calcium exchanger in cultured human retinal pigment epithelium.

    PubMed

    Mangini, N J; Haugh-Scheidt, L; Valle, J E; Cragoe, E J; Ripps, H; Kennedy, B G

    1997-12-01

    Regulation of intracellular free Ca2+ concentration ([Ca2+]i) by an Na+/Ca2+ exchanger was studied in cultures of human retinal pigment epithelial cells using Ca(2+)-indicator dyes (fura-2 and fluo-3) and digital fluorescence imaging. Mean resting [Ca2+]i of cultured RPE in a control Ringer solution was 189 +/- 16 nM. Replacing extracellular Na+ with N-methyl-D-glucamine elicited a two-fold rise in [Ca2+]i; the magnitude of the [Na+]o-free-induced rise in [Ca2+]i varied as a function of extracellular [Ca2+]. The [Na+]o-free response was not significantly affected by the Ca2+ channel blocker nifedipine, or by pretreatment with thapsigargin which depletes intracellular Ca2+ stores. By contrast, the [Na+]o-free-induced rise in [Ca2+]i was significantly reduced by CBDMB, an amiloride derivative that is highly selective for Na+/Ca2+ exchange inhibition. These findings indicate that removal of extracellular Na+ promotes net [Ca2+]i gain via Na+/Ca2+ exchange. Western and Northern blot analyses, respectively, confirmed the presence of Na+/Ca2+ exchanger protein and mRNA in cultures of human RPE. Specifically, Western blot analysis of whole cell lysates of cultured RPE using a polyclonal antibody made against the canine cardiac exchanger identified a major band at approximately 126 kD. Northern blot analysis of total human RPE RNA using a restriction fragment cRNA probe coding for the canine cardiac Na+/Ca2+ exchanger showed that the major exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-type Na+/Ca2+ exchanger in cultures of human RPE.

  10. The human corneal endothelium in keratoconus: A specular microscopic study.

    PubMed

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

    1979-10-01

    The corneal endothelium in 12 cases of keratoconus was examined with the clinical specular microscope. There appeared to be an increase in cellular pleomorphism with many cells considerably smaller than normal distributed throughout the endothelial cell population. There were also many large, elongated cells whose long axis showed a definite tendency to assume a similar directional orientation. The long axis of these cells seemed oriented toward the apex of the cone, and the cells appeared to have been stretched by the ectatic process. Many endothelial cells contained dark intracellular structures. Their significance is unknown. The single cornea in this series with a history of acute hydrops contained a localized area in which the endothelial cells were seven to ten times larger than normal. This suggests that rupture of the endothelium and Descemet's membrane, responsible for the acute edematous process, occurs at this site, and that the adjacent cells enlarged to fill the defect.

  11. Visualization of ex vivo human ciliated epithelium and induced flow using optical coherence tomography (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Ling, Yuye; Gamm, Uta A.; Yao, Xinwen; Arteaga-Solis, Emilio; Emala, Charles W.; Choma, Michael A.; Hendon, Christine P.

    2017-04-01

    The ciliated epithelium is important to the human respiratory system because it clears mucus that contains harmful microorganisms and particulate matter. We report the ex vivo visualization of human trachea/bronchi ciliated epithelium and induced flow characterized by using spectral-domain optical coherence tomography (SD-OCT). A total number of 17 samples from 7 patients were imaged. Samples were obtained from Columbia University Department of Anesthesiology's tissue bank. After excision, the samples were placed in Gibco Medium 199 solution with oxygen at 4°C until imaging. The samples were maintained at 36.7°C throughout the experiment. The imaging protocol included obtaining 3D volumes and 200 consecutive B-scans parallel to the head-to-feet direction (superior-inferior axis) of the airway, using Thorlabs Telesto system at 1300 nm at 28 kHz A-line rate and a custom built high resolution SDOCT system at 800nm at 32 kHz A-line rate. After imaging, samples were processed with H and E histology. Speckle variance of the time resolved datasets demonstrate significant contrast at the ciliated epithelium sites. Flow images were also obtained after injecting 10μm polyester beads into the solution, which shows beads traveling trajectories near the ciliated epithelium areas. In contrary, flow images taken in the orthogonal plane show no beads traveling trajectories. This observation is in line with our expectation that cilia drive flow predominantly along the superior-inferior axis. We also observed the protective function of the mucus, shielding the epithelium from the invasion of foreign objects such as microspheres. Further studies will be focused on the cilia's physiological response to environmental changes such as drug administration and physical injury.

  12. Distributions of elements in the human retinal pigment epithelium.

    PubMed

    Ulshafer, R J; Allen, C B; Rubin, M L

    1990-01-01

    Distributions of elements above the atomic number of sodium were mapped in the retinal pigment epithelia of eight human eyes. X-ray energy spectra and maps were collected from cryofixed, freeze-dried, and epoxy-embedded tissues using energy-dispersive x-ray microanalysis. All eyes had high concentrations of phosphorus in the nuclei of retinal pigment epithelial cells. Melanosomes were rich in sulfur, zinc, calcium, and iron. Lipofuscin and cytoplasm contained only phosphorus and sulfur in detectable amounts. Drusen, when present, contained phosphorus and calcium. Six eyes had a prominent aluminum peak recorded from melanosomes, nuclei, and Bruch's membrane. In one pair of 90-year-old eyes, small, electron-dense deposits surrounded many melanosomes and contained mercury and selenium. Retinal pigment epithelial melanosomes may bind and accumulate metals and other potentially toxic ions over time, preventing them from reaching the neural retina.

  13. An immunohistological study of cytokeratin 20 in human and mammalian oral epithelium.

    PubMed

    Barrett, A W; Cort, E M; Patel, P; Berkovitz, B K

    2000-10-01

    Cytokeratin (CK) 20 is a low molecular-weight intermediate filament reportedly expressed only by benign and malignant gastrointestinal epithelium, urothelium and Merkel cells. The main aims here were to map its expression in normal oral mucosa of humans and other mammals, and to determine whether it was expressed by abnormal human oral epithelium. Salivary and odontogenic epithelium were also analysed. An immunoperoxidase method was used on wax-embedded and cryostat sections. In addition, double-labelling experiments were undertaken to determine the association between CK 20 expression and that of CK 8/18 or S100 protein. Normal human oral mucosa from four sites, together with abdominal skin, was studied in autopsy samples from 32 individuals. CK 20-positive, basally situated, round or angular cells, consistent with Merkel cells, were recorded in 24/32 (75.0%) samples of mandibular gingiva, 25/32 (78.1%) samples of hard palate, 7/32 (21.9%) samples of buccal mucosa, 0/32 samples of lateral border of tongue, and 2/32 (6.3%) samples of abdominal skin. Double-labelling showed that all CK 20-positive Merkel cells also expressed CK 8/18 and S100. The only other cells to express CK 20 were human taste buds. There was no expression by dysplastic or invasive oral epithelium from biopsy samples. Colonic mucosa showed luminal-cell positivity in man, marmoset, ferret, rabbit and guinea-pig, but oral mucosa was universally negative in non-human species. It is concluded that in oral mucosa CK 20 is a specific marker of Merkel cells and taste buds, that Merkel cells are more frequently present in keratinized than non-keratinized oral mucosa, that CK 20-positive Merkel cells are also S100-positive, that there may be interspecies variations in CK 20 polypeptide composition and that, by contrast to urothelium, CK 20 has no value in the diagnosis of oral epithelial dysplasia.

  14. Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells.

    PubMed

    Ma, Yanling; Xu, Yongsheng; Xiao, Zhifeng; Yang, Wei; Zhang, Chun; Song, E; Du, Yiqin; Li, Lingsong

    2006-02-01

    To examine whether transplantation of human mesenchymal stem cells (MSCs) could reconstruct the corneal damage and also whether grafted MSCs could differentiate into corneal epithelial cells, we isolated MSCs from healthy donors. After growth and expansion on amniotic membrane, cells were transplanted into rat corneas 7 days after chemical burns. Reconstruction of the damaged cornea and the rat vision were measured once a week by slit lamp and by an optokinetic head-tracking instrument, respectively. Corneas were then cut out, fixed, and imbedded for immunofluorescent study of the expression of keratin 3 and keratin-pan as epithelial cell markers. Expression of CD45, interleukin 2, and metalloproteinase-2 was also investigated for inflammation and inflammation-related angiogenesis. The data showed that transplantation of MSCs, like limbal epithelial stem cells, successfully reconstructed damaged rat corneal surface. Interestingly, the therapeutic effect of the transplantation may be associated with the inhibition of inflammation and angiogenesis after transplantation of MSCs rather than the epithelial differentiation from MSCs. This study provides the first line of evidence that MSCs can be used for reconstruction of damaged corneas, presenting a new source for autotransplantation in the treatment of corneal disorders.

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

    PubMed Central

    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

  16. Corneal epithelial wound healing and bactericidal effect of conditioned medium from human uterine cervical stem cells.

    PubMed

    Bermudez, Maria A; Sendon-Lago, Juan; Eiro, Noemi; Treviño, Mercedes; Gonzalez, Francisco; Yebra-Pimentel, Eva; Giraldez, Maria Jesus; Macia, Manuel; Lamelas, Maria Luz; Saa, Jorge; Vizoso, Francisco; Perez-Fernandez, Roman

    2015-01-22

    To evaluate the effect of conditioned medium from human uterine cervical stem cells (CM-hUCESCs) on corneal epithelial healing in a rat model of dry eye after alkaline corneal epithelial ulcer. We also tested the bactericidal effect of CM-hUCESCs. Dry eye was induced in rats by extraocular lacrimal gland excision, and corneal ulcers were produced using NaOH. Corneal histologic evaluation was made with hematoxylin-eosin (H&E) staining. Real-time PCR was used to evaluate mRNA expression levels of proinflammatory cytokines. We also studied the bactericidal effect of CM-hUCESCs in vitro and on infected corneal contact lenses (CLs) using Escherichia coli and Staphylococcus epidermidis bacteria. In addition, in order to investigate proteins from CM-hUCESCs that could mediate these effects, we carried out a human cytokine antibody array. After injury, dry eyes treated with CM-hUCESCs significantly improved epithelial regeneration and showed reduced corneal macrophage inflammatory protein-1 alpha (MIP-1α) and TNF-α mRNA expression as compared to untreated eyes and eyes treated with culture medium or sodium hyaluronate ophthalmic drops. In addition, we found in CM-hUCESCs high levels of proteins, such as tissue inhibitors of metalloproteinases 1 and 2, fibroblast growth factor 6 and 7, urokinase receptor, and hepatocyte growth factor, that could mediate these effects. In vitro, CM-hUCESCs showed a clear bactericidal effect on both E. coli and S. epidermidis and CLs infected with S. epidermidis. Analyses of CM-hUCESCs showed elevated levels of proteins that could be involved in the bactericidal effect, such as the chemokine (C-X-C motif) ligands 1, 6, 8, 10, and the chemokine (C-C motif) ligands 5 and 20. Treatment with CM-hUCESCs improved wound healing of alkali-injured corneas and showed a strong bactericidal effect on CLs. Patients using CLs and suffering from dry eye, allergies induced by commercial solutions, or small corneal injuries could benefit from this treatment

  17. Current status of corneal xenotransplantation.

    PubMed

    Kim, Mee Kum; Hara, Hidetaka

    2015-11-01

    Corneal allo-transplantation is a well-established technique to treat corneal blindness. However, the limited availability of human donors demands the exploration of alternative treatments such as corneal xenotransplantation (e.g., pigs as donors) and bioengineered corneas. Since the first attempt of corneal xenotransplantation using a donor pig cornea in 1844, great advances have been made in the development of genetically-engineered pigs, effective immunosuppressive protocols and the establishment of guidelines for the conduction of clinical trials. We highlight immunological and physio-anatomical barriers of corneal xenotransplantation, recent progress of corneal xenotransplantation in non-human-primates studies, and regulatory guidelines to conduct clinical trials for corneal xenotransplantation.

  18. Corneal Penetrating Elastin-Like Polypeptide Carriers

    PubMed Central

    George, Eric M.; Mahdi, Fakhri; Logue, Omar C.; Robinson, Grant G.

    2016-01-01

    Abstract Purpose: Elastin-like polypeptide (ELP) is a bioengineered protein widely applied as a drug carrier due to its biocompatibility and amenability to modification with cell-penetrating peptides (CPPs) and therapeutic agents. The purpose of this study was to determine whether topically applied ELP or CPP-fused ELPs penetrate the corneal barrier. Methods: In vitro binding and cytotoxicity to human corneal epithelial (HCE) cells were determined for ELP or CPP-ELPs. Corneal binding, clearance, and penetration were assessed in a rabbit model following topical application of the fluorescently labeled proteins by quantitative fluorescence imaging and histology. Results: ELP bound to HCE cells in vitro, and binding/uptake was enhanced 2- to 3-fold by the addition of CPPs. When applied topically to rabbit eyes, ELP accumulated in the cornea at levels 7.4-fold higher than did an equivalent dose of immunoglobulin G. Both ELP and a CPP-ELP penetrated the corneal epithelium and were detectable in the stroma. Addition of CPPs to ELP, however, did not significantly enhance corneal uptake or penetration in vivo relative to ELP alone. The polypeptides cleared from the cornea over a period of 20–30 min after application, after which cornea levels reached a steady state of 15–30 μg/mL for up to 3 h. Conclusions: The ELP drug carrier can penetrate the corneal epithelium and accumulate in the stroma. Given its amenability for fusion to multiple types of therapeutic agents, ELP has the potential to serve as a drug carrier for topical ocular applications. PMID:26672799

  19. Comparison of corneal epitheliotrophic capacities among human platelet lysates and other blood derivatives

    PubMed Central

    Huang, Chien-Jung; Sun, Yi-Chen; Christopher, Karen; Pai, Amy Shih-I; Lu, Chia-Ju; Hu, Fung-Rong; Lin, Szu-Yuan; Chen, Wei-Li

    2017-01-01

    Purpose To evaluate the corneal epitheliotropic abilities of two commercialized human platelet lysates (HPLs) and to compare the results with other blood derivatives, including human peripheral serum (HPS) and bovine fetal serum (FBS). Methods In vitro, human corneal epithelial cells were incubated in various concentrations (0%, 3%, 5% and 10%) of blood derivatives. Two commercialized HPLs, including UltraGRO TM (Helios, Atlanta, GA) and PLTMax (Mill Creek, Rochester, MI), were tested and compared with HPS and FBS. Scratch-induced directional wounding assay was performed to evaluate cellular migration. MTS assay was used to evaluate cellular proliferation. Cellular differentiation was examined by scanning electron microscopy, inverted microscopy and transepithelial electrical resistance. Sprague-Dawley rats were used to evaluate the effects of the blood derivatives on corneal epithelial wound healing in vivo. Different blood derivatives were applied topically every 2 hours for 2 days after corneal epithelial debridement. The concentrations of epidermal growth factor (EGF), transforming growth factor -β1 (TGF-β1), fibronectin, platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, and hyaluronic acid in different blood derivatives were evaluated by enzyme-linked immunosorbent assay (ELISA). Results In vitro experiments demonstrated statistically comparable epitheliotropic characteristics in cellular proliferation, migration, and differentiation for the two commercialized HPLs compared to FBS and HPS. Cells cultured without any serum were used as control group. The epitheliotropic capacities were statistically higher in the two commercialized HPLs compared to the control group (p<0.05). Among the different concentrations of blood derivatives, the preparations with 3% yielded better outcomes compared to 5% and 10%. In rats, HPLs also caused improved but not statistically significant wound healing compared to HPS. All the blood derivatives had better wound healing

  20. Translational label-free nonlinear imaging biomarkers to classify the human corneal microstructure

    PubMed Central

    Lombardo, Marco; Merino, David; Loza-Alvarez, Pablo; Lombardo, Giuseppe

    2015-01-01

    Diseases that affect the cornea can lead to severe vision loss and have tremendous social impact. These diseases are associated to deviations from normal structural order and orientation of collagen fibril bundles. Unfortunately, resolving non-invasively the corneal collagen structure is not possible to date. In this work, polarization sensitive second harmonic generation (pSHG) microscopy is used to obtain information with molecular specificity on microstructure of human corneas. This information is used to develop a set of label-free imaging biomarkers that were generated by means of a novel methodology based on mathematical tensorial calculus. The method is proven to be highly sensitive and robust. The use of these biomarkers permits accurate characterization of the anisotropic, depth-dependent, structural organization of corneal collagen fibril bundles without any a priori information. The method can be valuable to improve understanding of microstructural pathophysiological changes of the human cornea close to in vivo conditions. PMID:26309745

  1. A three-dimensional study of human fetal endocervix with special reference to its epithelium.

    PubMed

    Barberini, F; Makabe, S; Motta, P M

    1998-07-01

    The development of human fetal cervix has been systematically studied by SEM, obtaining a detailed map of its fine structure, particularly concerning the differentiation and maturation of the endocervical epithelium, including its "eversion" and "squamous metaplasia", normally occurring in postnatal life, but not yet observed in detail by electron microscopy in the fetus. Cervices from spontaneous abortion at 12, 15, 18, 20, 21 and 22 weeks and from intrauterine fetal death (hydrocephalus) at 31 weeks of development have been examined. At 12-15 weeks, as the canalization of the cervix proceeded, the endocervical epithelium consisted of high polyhedral cells, with regularly flattened or concave apices exhibiting scarce microvilli and often single primary cilia. Some narrow intercellular infoldings probably corresponded to primordial tubular glands. At the 18th week the epithelium was made up of a mosaic of flat or slightly raised polygonal cells, whose apical surface showed thin microplicae. At the 20th week a pseudostratified epithelium with many apically convex cells lined the cervical canal and the tubular glands. At 21 and 22 weeks "plicae palmatae" developed, covered by cells, often showing a smooth central area surrounded by microvilli, provided with a primary cilium and swollen by secretory material. This also formed rounded masses on the epithelium. In the lower part of the endocervix some very elongated cells showed short microplicae resulting from fusion of microvilli. At the 31st week secretion increased and its products spreading from the bottom of the glands contacted isolated ciliated cells at their openings and diffusely covered the surface epithelium. Most of the ectocervix exhibited squamous elements, with well-developed labyrinthine microplicae. These cells could overlap each other and also desquamate. The zone of the portio vaginalis around the os of the cervical canal appeared infolded and hypertrophic. Here, an indented squamo-columnar junction

  2. Chronic exposure to the ultraviolet radiation levels from arc welding does not result in obvious damage to the human corneal endothelium.

    PubMed

    Oblak, Emil; Doughty, Michael J

    2002-11-01

    Occupational exposure of the cornea to ultraviolet radiation (UVR, e.g. in welding) is a well-known cause of 'arc eye' (photo-keratoconjunctivitis), but has also been considered to be a risk for the development of alterations in the size (polymegethism) and shape (pleomorphism) of the deeper-lying human corneal endothelial cells. Human data are however limited and so a further study was undertaken, with a control group. Non-contact specular micrographs of the central region of the corneal endothelium were obtained from 40 white males aged between 32 and 63 years; 20 were arc welders with an average of 25 +/- 7 years job experience, while the others were office workers (n = 20). All the welders reported occupational exposure to UVR (i.e. welders 'flashes') and up to 3 times per year. None of the subjects had a history of contact lens wear, major eye disease or surgery. The endothelial image was scanned, projected onto an overlay and cell border marking carried out in a masked fashion. The overlay was independently analysed, by a customised semi-automated method, providing cell-border-adjusted data on cell areas and cell shape (sides) on 124 to 260 cells per image. The endothelial cell density (ECD) values were also calculated from individual cell area values. All corneas appeared to be healthy, and showed no fluorescein staining indicating damage to the surface epithelium. Central corneal thickness values were normal at 0.531 +/- 0.031 (mean +/- SD) and 0.527 +/- 0.036 mm in the welders and non-welders respectively. All endothelia appeared healthy, with no evidence of cell oedema. The group-mean endothelial cell area was 393 +/- 35 and 392 +/- 21 microm2, ECD values were 2855 +/- 224 cells mm(-2) and 2852 +/- 210 cells mm(-2), while the percentages of 6-sided cells were 60 +/- 5.2 and 59 +/- 4.1% respectively. Cell area distributions were statistically identical (p > or = 0.8), and cell area-side relationships were marginally, but not statistically different. This

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

    PubMed

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

    2014-01-01

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

  4. NGF and NGF-receptor expression of cultured immortalized human corneal endothelial cells

    PubMed Central

    Sornelli, Federica; Lambiase, Alessandro; Mantelli, Flavio

    2010-01-01

    Purpose Several growth factors, including nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), play an important role in the homeostasis of the ocular surface. The involvement of both these growth factors in the pathophysiology of intraocular tissues has been extensively investigated. Despite the expression of NGF receptors by corneal endothelium, to date the role of NGF on the endothelial cell remains to be determined. Using a clonal cell line of human corneal endothelial cells, the aim of this study was to investigate the expression of the NGF-receptor and the potential partnership of NGF and VEGF in maintaining cell viability in vitro. Methods A human endothelial cell line (B4G12), was cultured under serum-free conditions as previously described with and without addition of different concentrations of NGF, anti-NGF-antibody (ANA), or VEGF for 4 days and these cells were used for immuno-istochemical, biochemical, and molecular analyses. Results NGF induces overexpression of NGF-receptors and synthesis and release of VEGF by endothelial cells and these cells are able to produce and secrete NGF. Conclusions These observations indicate that human corneal endothelial cells are receptive to the action of NGF and that these cells may regulate NGF activity through autocrine/paracrine mechanisms. PMID:20680101

  5. Lactobacillus reuteri Inhibition of Enteropathogenic Escherichia coli Adherence to Human Intestinal Epithelium

    PubMed Central

    Walsham, Alistair D. S.; MacKenzie, Donald A.; Cook, Vivienne; Wemyss-Holden, Simon; Hews, Claire L.; Juge, Nathalie; Schüller, Stephanie

    2016-01-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal infant death in developing countries, and probiotic bacteria have been shown to provide health benefits in gastrointestinal infections. In this study, we have investigated the influence of the gut symbiont Lactobacillus reuteri on EPEC adherence to the human intestinal epithelium. Different host cell model systems including non-mucus-producing HT-29 and mucus-producing LS174T intestinal epithelial cell lines as well as human small intestinal biopsies were used. Adherence of L. reuteri to HT-29 cells was strain-specific, and the mucus-binding proteins CmbA and MUB increased binding to both HT-29 and LS174T cells. L. reuteri ATCC PTA 6475 and ATCC 53608 significantly inhibited EPEC binding to HT-29 but not LS174T cells. While pre-incubation of LS174T cells with ATCC PTA 6475 did not affect EPEC attaching/effacing (A/E) lesion formation, it increased the size of EPEC microcolonies. ATCC PTA 6475 and ATCC 53608 binding to the mucus layer resulted in decreased EPEC adherence to small intestinal biopsy epithelium. Our findings show that L. reuteri reduction of EPEC adhesion is strain-specific and has the potential to target either the epithelium or the mucus layer, providing further rationale for the selection of probiotic strains. PMID:26973622

  6. Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity

    PubMed Central

    2013-01-01

    Background As the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype. Methods To generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed. Results We successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process. Conclusion Development of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents. PMID:24298994

  7. Magnetic Field-Guided Cell Delivery with Nanoparticle-Loaded Human Corneal Endothelial Cells

    PubMed Central

    Moysidis, Stavros N.; Alvarez-Delfin, Karen; Peschansky, Veronica J.; Salero, Enrique; Weisman, Alejandra D.; Bartakova, Alena; Raffa, Gabriella A.; Merkhofer, Richard M.; Kador, Karl E.; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

    2015-01-01

    To improve the delivery and integration of cell therapy using magnetic cell guidance for replacement of corneal endothelium, here we assess magnetic nanoparticles’ (MNPs) effects on human corneal endothelial cells (HCECs) in vitro. Biocompatible, 50 nm superparamagnetic nanoparticles endocytosed by cultured HCECs induced no short- or long-term change in viability or identity. Assessment of guidance of the magnetic HCECs in the presence of different magnet shapes and field strengths showed a 2.4-fold increase in delivered cell density compared to gravity alone. After cell delivery, HCECs formed a functional monolayer, with no difference in tight junction formation between MNP-loaded and control HCECs. These data suggest that nanoparticle-mediated magnetic cell delivery may increase the efficiency of cell delivery without compromising HCEC survival, identity or function. Future studies may assess the safety and efficacy of this therapeutic modality in vivo. PMID:25596075

  8. Dose dependent cytotoxicity of pranoprofen in cultured human corneal endothelial cells by inducing apoptosis.

    PubMed

    Li, Yi-Han; Wen, Qian; Fan, Ting-Jun; Ge, Yuan; Yu, Miao-Miao; Sun, Ling-Xiao; Zhao, Yu

    2015-01-01

    Pranoprofen (PPF), a non-steroidal anti-inflammatory drugs (NSAIDs), is often used in keratitis treatment in clinic. Several studies have assessed in vitro the cytotoxicity of topical NSAIDs to corneal epithelial cells due to its importance for predicting human corneal toxicity. Damage by cytotoxic drugs can result in excessive loss of human corneal endothelial (HCE) cells which lead to decompensation of the endothelium and eventual loss of visual acuity. However, the endothelial cytotoxicity of PPF has not yet been reported using an in vitro model of HCE cells. This study assessed the cytotoxicity of PPF to HCE cells and its underlying mechanism. Cellular viability was determined using inverted phase contrast light microscopy, and plasma membrane permeability, genomic DNA fragmentation, and ultrastructure were detected by acridine orange/ethidium bromide staining, DNA agarose gel electrophoresis, and transmission electron microscopy (TEM), respectively. The results on cellular viability showed that PPF at concentrations ranging from 0.0625 to 1.0 g/l had poignant cytotoxicity to HCE cells, and the extent of its cytotoxicity was dose- and time-dependent. Further characterization indicated that PPF induced plasma membrane permeability elevation, DNA fragmentation, and apoptotic body formation, proving its apoptosis inducing effect on HCE cells. In conclusion, PPF above 0.0625 g/l has poignant cytotoxicity on HCE cells in vitro by inducing cell apoptosis, and should be carefully employed in eye clinic.

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

    PubMed

    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.

  10. Curvature sensor for the measurement of the static corneal topography and the dynamic tear film topography in the human eye

    NASA Astrophysics Data System (ADS)

    Gruppetta, Steve; Koechlin, Laurent; Lacombe, François; Puget, Pascal

    2005-10-01

    A system to measure the topography of the first optical surface of the human eye noninvasively by using a curvature sensor is described. The static corneal topography and the dynamic topography of the tear film can both be measured, and the topographies obtained are presented. The system makes possible the study of the dynamic aberrations introduced by the tear film to determine their contribution to the overall ocular aberrations in healthy eyes, eyes with corneal pathologies, and eyes wearing contact lenses.

  11. Multiscale Investigation of the Depth-Dependent Mechanical Anisotropy of the Human Corneal Stroma

    PubMed Central

    Labate, Cristina; Lombardo, Marco; De Santo, Maria P.; Dias, Janice; Ziebarth, Noel M.; Lombardo, Giuseppe

    2015-01-01

    Purpose. To investigate the depth-dependent mechanical anisotropy of the human corneal stroma at the tissue (stroma) and molecular (collagen) level by using atomic force microscopy (AFM). Methods. Eleven human donor corneas were dissected at different stromal depths by using a microkeratome. Mechanical measurements were performed in 15% dextran on the surface of the exposed stroma of each sample by using a custom-built AFM in force spectroscopy mode using both microspherical (38-μm diameter) and nanoconical (10-nm radius of curvature) indenters at 2-μm/s and 15-μm/s indentation rates. Young's modulus was determined by fitting force curve data using the Hertz and Hertz-Sneddon models for a spherical and a conical indenter, respectively. The depth-dependent anisotropy of stromal elasticity was correlated with images of the corneal stroma acquired by two-photon microscopy. Results. The force curves were obtained at stromal depths ranging from 59 to 218 μm. At the tissue level, Young's modulus (ES) showed a steep decrease at approximately 140-μm stromal depth (from 0.8 MPa to 0.3 MPa; P = 0.03) and then was stable in the posterior stroma. At the molecular level, Young's modulus (EC) was significantly greater than at the tissue level; EC decreased nonlinearly with increasing stromal depth from 3.9 to 2.6 MPa (P = 0.04). The variation of microstructure through the thickness correlated highly with a nonconstant profile of the mechanical properties in the stroma. Conclusions. The corneal stroma exhibits unique anisotropic elastic behavior at the tissue and molecular levels. This knowledge may benefit modeling of corneal behavior and help in the development of biomimetic materials. PMID:26098472

  12. Analysis of damage to human ciliated nasopharyngeal epithelium by Neisseria meningitidis.

    PubMed Central

    Stephens, D S; Whitney, A M; Melly, M A; Hoffman, L H; Farley, M M; Frasch, C E

    1986-01-01

    We used an in vitro model of human nasopharyngeal tissue in organ culture to evaluate the effects of Neisseria meningitidis on human cilia and ciliary function. Encapsulated, viable meningococci damaged ciliated epithelium of nasopharyngeal organ cultures, whereas Neisseria subflava, a commensal species, did not. Meningococcus-induced ciliary damage was due to loss of ciliated cells to which meningococci were not attached. Damage was seen with piliated and nonpiliated meningococci and did not appear to require the presence of other specific meningococcal surface proteins. Meningococcal viability was a requirement for both ciliary damage and interactions of meningococci with microvilli of nonciliated epithelial cells. That is, filter-sterilized supernatants from meningococcus-infected organ cultures, heat-killed meningococci at high inoculum, and purified meningococcal or gonococcal lipopolysaccharide at concentrations of 100 micrograms/ml did not damage ciliary activity of nasopharyngeal organ cultures. In contrast, meningococcal lipopolysaccharide at 10 micrograms/ml markedly damaged ciliary activity of human fallopian tube organ cultures, suggesting a selective toxicity of lipopolysaccharide for specific human ciliated cells. Damage to nasopharyngeal ciliated epithelium by N. meningitidis may be an important first step in meningococcal colonization of the human nasopharynx, but meningococcal lipopolysaccharide does not appear to be directly responsible for this toxicity. Images PMID:2867973

  13. Morphologic alterations in the epithelium of the human oviduct induced by a low dosis gestagen.

    PubMed

    Spornitz, U M; Ludwig, K S; Mall-Haefeli, M

    1977-11-29

    Morphological alterations in the epithelium of the human oviduct related to continuous administration of low dosage lynestrenol were studied in 10 women by electron microscopy. The effect of lynestrenol on the morphology of the tubal epithelium was shown to be time dependent. First the discharge of secretory products from the non-ciliated cells was inhibited. Secondary to this the synthetic activities of the cells were drastically decreased. This inactivation of the tubal epithelium reached its maximum after 6 months. At this time the epithelial cells, particularly the non-ciliated cells were almost devoid of organelles and had an electron transparent cytoplasm. In addition to the action of lynestrenol on the secretory and synthetic activities of the cells a strong ciliogenetic effect could be observed after about 3 months of lynestrenol administration. The ratio of ciliated to non-ciliated cells changed from 1:1 at the beginning of the treatment to 2.9:1 after 6 months. This ratio was then maintained and could still be found in patients who had used lynestrenol for 25 and 39 months. In contrast to the persistent effect of lynestrenol on the ciliary apparatus of the cells the synthetic activities and the secretory mechanism were partially restored with long standing use. The importance of the observed changes and their possible participation in the contraceptive action of lynestrenol are discussed.

  14. Growth factors and corneal endothelial cells: III. Stimulation of adult human corneal endothelial cell mitosis in vitro by defined mitogenic agents.

    PubMed

    Schultz, G; Cipolla, L; Whitehouse, A; Eiferman, R; Woost, P; Jumblatt, M

    1992-01-01

    Human corneal endothelial cells (HCEC) do not mitose extensively in vivo after damage to the endothelial layer. However, HCEC will divide in vitro if cultured under appropriate conditions. We measured the ability of various sera, plasma, growth factors, and nutritional substances to stimulate mitosis of HCEC during 5 days of organ culture after a central freeze injury to the endothelium. Supplementation of a chemically defined medium (CDM) with 20% fetal human serum (FHS) induced significantly higher numbers of mitotic figures or labeled nuclei of human or cat corneas compared with paired corneas cultured in CDM alone. Furthermore, addition of 20% FHS produced more labeled nuclei than did addition of 20% fetal bovine serum or 20% adult human serum. Dialyzed fetal human serum failed to stimulate mitosis, indicating that one or more components of fetal human serum with molecular weight less than 12,000 are essential for mitosis. Human plasma also failed to stimulate mitosis, but an extract of human platelets significantly stimulated high levels of nuclear labeling, suggesting that growth factors contained in platelet granules were responsible for serum-stimulated mitosis of HCEC. Addition of 100 nM epidermal growth factor (EGF) or 10 microM insulin to CDM supplemented with low levels of adult human serum (0.5%) stimulated significantly higher numbers of labeled nuclei compared with paired corneas cultured with 0.5% adult human serum. Supplementation of corneal storage media (K-Sol and CSM) with a mixture of chemically defined agents consisting of EGF, insulin, transferrin, selenium, linoleic acid, and albumin stimulated significantly higher numbers of labeled nuclei compared with paired corneas cultured in the unsupplemented corneal storage media.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Structural differentiation of human uterine luminal and glandular epithelium during early pregnancy: an ultrastructural and immunohistochemical study.

    PubMed

    Demir, R; Kayisli, U A; Celik-Ozenci, C; Korgun, E T; Demir-Weusten, A Y; Arici, A

    2002-01-01

    The differentiation of human endometrial epithelium is a dynamic event that occurs throughout the menstrual cycle and early pregnancy. The structural transformation and differentiation of human uterine luminal and glandular epithelium of early human pregnancy (n=14) was investigated ultrastructurally and immunohistochemically using antibodies against cytokeratin (CT), endothelial marker CD31, Fas, and proliferating cell nuclear antigen (PCNA). Ultrastructurally, luminal epithelial cells showed distinctive euchromatic nuclei with prominent nucleoli and relatively loose cell membranes in all poles (apical to basal). Subcellular components were easily recognized in luminal epithelium except in degenerating cells. Mainly two cell types, dark and clear cells, formed the glandular epithelium. In the early gestation period, microvilli were abundant on the apical and apico-lateral poles of these cells. Only a few cytoplasmic projections were observed in dark cells. Numerous cilia were observed on the apical pole of some clear cells, located at the adluminal segment. In contrast, dark cells lacked cilia, nuclear channels, or giant mitochondrial profiles. Glycogen synthesis and apocrine secretion were recognizable for several days during early gestation. The apocrine secretory activity differed among dark cells of the glandular epithelium. The immunoreactivity of PCNA and Fas, and ultrastructural observations in the glandular epithelium suggest that, even in different segments of the same gland, epithelial cells do not regress during early gestation, but proliferate, perhaps representing a resistance against trophoblastic invasion. These morphological and molecular changes suggest that both luminal and glandular epithelium may play an important role in cellular defense and limitation for trophoblastic invasion during early pregnancy since plasma membrane alterations of the surface epithelium take place at the apical, basal and lateral poles compared to early secretory phase

  16. Human cadaver retina model for retinal heating during corneal surgery with a femtosecond laser

    NASA Astrophysics Data System (ADS)

    Sun, Hui; Fan, Zhongwei; Yun, Jin; Zhao, Tianzhuo; Yan, Ying; Kurtz, Ron M.; Juhasz, Tibor

    2014-02-01

    Femtosecond lasers are widely used in everyday clinical procedures to perform minimally invasive corneal refractive surgery. The intralase femtosecond laser (AMO Corp. Santa Ana, CA) is a common example of such a laser. In the present study a numerical simulation was developed to quantify the temperature rise in the retina during femtosecond intracorneal surgery. Also, ex-vivo retinal heating due to laser irradiation was measured with an infrared thermal camera (Fluke Corp. Everett, WA) as a validation of the simulation. A computer simulation was developed using Comsol Multiphysics to calculate the temperature rise in the cadaver retina during femtosecond laser corneal surgery. The simulation showed a temperature rise of less than 0.3 degrees for realistic pulse energies for the various repetition rates. Human cadaver retinas were irradiated with a 150 kHz Intralase femtosecond laser and the temperature rise was measured withan infrared thermal camera. Thermal camera measurements are in agreement with the simulation. During routine femtosecond laser corneal surgery with normal clinical parameters, the temperature rise is well beneath the threshold for retina damage. The simulation predictions are in agreement with thermal measurements providing a level of experimental validation.

  17. Focal adhesion kinase knockdown modulates the response of human corneal epithelial cells to topographic cues.

    PubMed

    Dreier, Britta; Raghunathan, Vijaya Krishna; Russell, Paul; Murphy, Christopher J

    2012-12-01

    A rapidly expanding literature broadly documents the impact of biophysical cues on cellular behaviors. In spite of increasing research efforts in this field, the underlying signaling processes are poorly understood. One of the candidate molecules for being involved in mechanotransduction is focal adhesion kinase (FAK). To examine the role of FAK in the response of immortalized human corneal epithelial (hTCEpi) cells to topographic cues, FAK was depleted by siRNA transfection. Contrary to expectations, FAK knockdown resulted in an enhanced response with a greater number of hTCEpi cells aligned to the long axis of anisotropically ordered surface ridges and grooves. Both underlying topographic features and FAK depletion modulated the migration of corneal epithelial cells. The impact of FAK knockdown on both migration and alignment varied depending on the topographic cues to which the cells were exposed, with the most significant change observed on the biologically relevant size scale (400nm). Additionally, a change in expression of genes encoding perinuclear Nesprins 1 and 2 (SYNE1, 2) was observed in response to topographic cues. SYNE1/2 expression was also altered by FAK depletion, suggesting that these proteins might represent a link between cytosolic and nuclear signaling processes. The data presented here have relevance to our understanding of the fundamental processes involved in corneal cell behavior to topographic cues. These results highlight the importance of incorporating biophysical cues in the conduction of in vitro studies and into the design and fabrication of implantable prosthetics.

  18. The conversion of delta 5-steriods to testosterone and androstenedione in human amniotic epithelium in vitro.

    PubMed

    Sulcová, J; Jirásek, J E; Stárka, L

    1977-09-01

    3 beta-Hydroxysteroid dehydrogenase / delta 5-4 isomerase activity was demonstrated in the human amniotic epithelium from the first trimester of pregnancy. The evidence was based on the in vitro formation of [4-14C] testosterone and [4-14C] androstenedione from [4-14C] 5-androstenediol and [4-14CA1 DEHYDROEPIANDROSTERONE, RESPECTIVELY. The activity of the enzyme studied in age dependent, reaching a maximum in the 8th-9th week of pregnancy and decreasing to negligible values at the end of the second trimester of gestation.

  19. Corneal Regeneration After Photorefractive Keratectomy: A Review☆

    PubMed Central

    Tomás-Juan, Javier; Murueta-Goyena Larrañaga, Ane; Hanneken, Ludger

    2014-01-01

    Photorefractive keratectomy (PRK) remodels corneal stroma to compensate refractive errors. The removal of epithelium and the ablation of stroma provoke the disruption of corneal nerves and a release of several peptides from tears, epithelium, stroma and nerves. A myriad of cytokines, growth factors, and matrix metalloproteases participate in the process of corneal wound healing. Their balance will determine if reepithelization and stromal remodeling are appropriate. The final aim is to achieve corneal transparency for restoring corneal function, and a proper visual quality. Therefore, wound-healing response is critical for a successful refractive surgery. Our goal is to provide an overview into how corneal wounding develops following PRK. We will also review the influence of intraoperative application of mitomycin C, bandage contact lenses, anti-inflammatory and other drugs in preventing corneal haze and post-PRK pain. PMID:25444646

  20. Corneal Regeneration After Photorefractive Keratectomy: A Review.

    PubMed

    Tomás-Juan, Javier; Murueta-Goyena Larrañaga, Ane; Hanneken, Ludger

    2015-01-01

    Photorefractive keratectomy (PRK) remodels corneal stroma to compensate refractive errors. The removal of epithelium and the ablation of stroma provoke the disruption of corneal nerves and a release of several peptides from tears, epithelium, stroma and nerves. A myriad of cytokines, growth factors, and matrix metalloproteases participate in the process of corneal wound healing. Their balance will determine if reepithelization and stromal remodeling are appropriate. The final aim is to achieve corneal transparency for restoring corneal function, and a proper visual quality. Therefore, wound-healing response is critical for a successful refractive surgery. Our goal is to provide an overview into how corneal wounding develops following PRK. We will also review the influence of intraoperative application of mitomycin C, bandage contact lenses, anti-inflammatory and other drugs in preventing corneal haze and post-PRK pain.

  1. Electrogenic transport and K(+) ion channel expression by the human endolymphatic sac epithelium.

    PubMed

    Kim, Sung Huhn; Kim, Bo Gyung; Kim, Jin Young; Roh, Kyung Jin; Suh, Michelle J; Jung, JinSei; Moon, In Seok; Moon, Sung K; Choi, Jae Young

    2015-12-14

    The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K(+) channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K(+) channels in the electrogenic transport of human ES epithelium. The identified K(+) channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K(+) transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid.

  2. Electrogenic transport and K+ ion channel expression by the human endolymphatic sac epithelium

    PubMed Central

    Kim, Sung Huhn; Kim, Bo Gyung; Kim, Jin Young; Roh, Kyung Jin; Suh, Michelle J.; Jung, JinSei; Moon, In Seok; Moon, Sung K.; Choi, Jae Young

    2015-01-01

    The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K+ channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K+ channels in the electrogenic transport of human ES epithelium. The identified K+ channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K+ transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid. PMID:26655723

  3. Oxygen-deficient metabolism and corneal edema.

    PubMed

    Leung, B K; Bonanno, J A; Radke, C J

    2011-11-01

    Wear of low-oxygen-transmissible soft contact lenses swells the cornea significantly, even during open eye. Although oxygen-deficient corneal edema is well-documented, a self-consistent quantitative prediction based on the underlying metabolic reactions is not available. We present a biochemical description of the human cornea that quantifies hypoxic swelling through the coupled transport of water, salt, and respiratory metabolites. Aerobic and anaerobic consumption of glucose, as well as acidosis and pH buffering, are incorporated in a seven-layer corneal model (anterior chamber, endothelium, stroma, epithelium, postlens tear film, contact lens, and prelens tear film). Corneal swelling is predicted from coupled transport of water, dissolved salts, and especially metabolites, along with membrane-transport resistances at the endothelium and epithelium. At the endothelium, the Na+/K+ - ATPase electrogenic channel actively transports bicarbonate ion from the stroma into the anterior chamber. As captured by the Kedem-Katchalsky membrane-transport formalism, the active bicarbonate-ion flux provides the driving force for corneal fluid pump-out needed to match the leak-in tendency of the stroma. Increased lactate-ion production during hypoxia osmotically lowers the pump-out rate requiring the stroma to swell to higher water content. Concentration profiles are predicted for glucose, water, oxygen, carbon dioxide, and hydronium, lactate, bicarbonate, sodium, and chloride ions, along with electrostatic potential and pressure profiles. Although the active bicarbonate-ion pump at the endothelium drives bicarbonate into the aqueous humor, we find a net flux of bicarbonate ion into the cornea that safeguards against acidosis. For the first time, we predict corneal swelling upon soft-contact-lens wear from fundamental biophysico-chemical principles. We also successfully predict that hypertonic tear alleviates contact-lens-induced edema.

  4. Oxygen-deficient metabolism and corneal edema

    PubMed Central

    Leung, B.K.; Bonanno, J.A.; Radke, C.J.

    2014-01-01

    Wear of low-oxygen-transmissible soft contact lenses swells the cornea significantly, even during open eye. Although oxygen-deficient corneal edema is well-documented, a self-consistent quantitative prediction based on the underlying metabolic reactions is not available. We present a biochemical description of the human cornea that quantifies hypoxic swelling through the coupled transport of water, salt, and respiratory metabolites. Aerobic and anaerobic consumption of glucose, as well as acidosis and pH buffering, are incorporated in a seven-layer corneal model (anterior chamber, endothelium, stroma, epithelium, postlens tear film, contact lens, and prelens tear film). Corneal swelling is predicted from coupled transport of water, dissolved salts, and especially metabolites, along with membrane-transport resistances at the endothelium and epithelium. At the endothelium, the Na+/K+ - ATPase electrogenic channel actively transports bicarbonate ion from the stroma into the anterior chamber. As captured by the Kedem–Katchalsky membrane-transport formalism, the active bicarbonate-ion flux provides the driving force for corneal fluid pump-out needed to match the leak-in tendency of the stroma. Increased lactate-ion production during hypoxia osmotically lowers the pump-out rate requiring the stroma to swell to higher water content. Concentration profiles are predicted for glucose, water, oxygen, carbon dioxide, and hydronium, lactate, bicarbonate, sodium, and chloride ions, along with electrostatic potential and pressure profiles. Although the active bicarbonate-ion pump at the endothelium drives bicarbonate into the aqueous humor, we find a net flux of bicarbonate ion into the cornea that safeguards against acidosis. For the first time, we predict corneal swelling upon soft-contact-lens wear from fundamental biophysico-chemical principles. We also successfully predict that hypertonic tear alleviates contact-lens-induced edema. PMID:21820076

  5. Microarray analysis of cell cycle gene expression in adult human corneal endothelial cells.

    PubMed

    Ha Thi, Binh Minh; Campolmi, Nelly; He, Zhiguo; Pipparelli, Aurélien; Manissolle, Chloé; Thuret, Jean-Yves; Piselli, Simone; Forest, Fabien; Peoc'h, Michel; Garraud, Olivier; Gain, Philippe; Thuret, Gilles

    2014-01-01

    Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts.

  6. Microarray Analysis of Cell Cycle Gene Expression in Adult Human Corneal Endothelial Cells

    PubMed Central

    Ha Thi, Binh Minh; Campolmi, Nelly; He, Zhiguo; Pipparelli, Aurélien; Manissolle, Chloé; Thuret, Jean-Yves; Piselli, Simone; Forest, Fabien; Peoc'h, Michel; Garraud, Olivier; Gain, Philippe; Thuret, Gilles

    2014-01-01

    Corneal endothelial cells (ECs) form a monolayer that controls the hydration of the cornea and thus its transparency. Their almost nil proliferative status in humans is responsible, in several frequent diseases, for cell pool attrition that leads to irreversible corneal clouding. To screen for candidate genes involved in cell cycle arrest, we studied human ECs subjected to various environments thought to induce different proliferative profiles compared to ECs in vivo. Donor corneas (a few hours after death), organ-cultured (OC) corneas, in vitro confluent and non-confluent primary cultures, and an immortalized EC line were compared to healthy ECs retrieved in the first minutes of corneal grafts. Transcriptional profiles were compared using a cDNA array of 112 key genes of the cell cycle and analysed using Gene Ontology classification; cluster analysis and gene map presentation of the cell cycle regulation pathway were performed by GenMAPP. Results were validated using qRT-PCR on 11 selected genes. We found several transcripts of proteins implicated in cell cycle arrest and not previously reported in human ECs. Early G1-phase arrest effectors and multiple DNA damage-induced cell cycle arrest-associated transcripts were found in vivo and over-represented in OC and in vitro ECs. Though highly proliferative, immortalized ECs also exhibited overexpression of transcripts implicated in cell cycle arrest. These new effectors likely explain the stress-induced premature senescence that characterizes human adult ECs. They are potential targets for triggering and controlling EC proliferation with a view to increasing the cell pool of stored corneas or facilitating mass EC culture for bioengineered endothelial grafts. PMID:24747418

  7. Reconstituted Human Upper Airway Epithelium as 3-D In Vitro Model for Nasal Polyposis

    PubMed Central

    de Borja Callejas, Francisco; Martínez-Antón, Asunción; Alobid, Isam; Fuentes, Mireya; Cortijo, Julio; Picado, César

    2014-01-01

    Background Primary human airway epithelial cells cultured in an air-liquid interface (ALI) develop a well-differentiated epithelium. However, neither characterization of mucociliar differentiation overtime nor the inflammatory function of reconstituted nasal polyp (NP) epithelia have been described. Objectives 1st) To develop and characterize the mucociliar differentiation overtime of human epithelial cells of chronic rhinosinusitis with nasal polyps (CRSwNP) in ALI culture system; 2nd) To corroborate that 3D in vitro model of NP reconstituted epithelium maintains, compared to control nasal mucosa (NM), an inflammatory function. Methods Epithelial cells were obtained from 9 NP and 7 control NM, and differentiated in ALI culture for 28 days. Mucociliary differentiation was characterized at different times (0, 7, 14, 21, and 28 days) using ultrastructure analysis by electron microscopy; ΔNp63 (basal stem/progenitor cell), β-tubulin IV (cilia), and MUC5AC (goblet cell) expression by immunocytochemistry; and mucous (MUC5AC, MUC5B) and serous (Lactoferrin) secretion by ELISA. Inflammatory function of ALI cultures (at days 0, 14, and 28) through cytokine (IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70) and chemokine (RANTES, MIG, MCP-1, IP-10, eotaxin-1, and GM-CSF) production was analysed by CBA (Cytometric Bead Array). Results In both NP and control NM ALI cultures, pseudostratified epithelium with ciliated, mucus-secreting, and basal cells were observed by electron microscopy at days 14 and 28. Displaying epithelial cell re-differentation, β-tubulin IV and MUC5AC positive cells increased, while ΔNp63 positive cells decreased overtime. No significant differences were found overtime in MUC5AC, MUC5B, and lactoferrin secretions between both ALI cultures. IL-8 and GM-CSF were significantly increased in NP compared to control NM regenerated epithelia. Conclusion Reconstituted epithelia from human NP epithelial cells cultured in ALI system provides a 3D in vitro model

  8. Staphylococcus aureus triggers nitric oxide production in human upper airway epithelium

    PubMed Central

    Carey, Ryan M.; Workman, Alan D.; Chen, Bei; Adappa, Nithin D.; Palmer, James N.; Kennedy, David W.; Lee, Robert J.; Cohen, Noam A.

    2016-01-01

    Background Nitric oxide (NO) is an important antibacterial defense molecule produced by upper airway (sinonasal) epithelial cells. We previously showed that a bitter taste receptor expressed in airway epithelium detects quorum-sensing molecules secreted by Gram-negative bacteria and subsequently triggers bactericidal NO production. We hypothesized that the upper airway epithelium may also be able to detect the Gram-positive aerobe Staphylococcus aureus and mount an NO response. Methods Human sinonasal air-liquid interface (ALI) cultures were treated with methicillin-resistant S. aureus (MRSA)-conditioned medium (CM), and NO production was measured using fluorescence imaging. Inhibitors of bitter taste receptor signaling were used to pharmacologically determine if this pathway was involved in the production of NO. Results A low-molecular-weight, heat, and protease-stabile product found in MRSA CM induced differential, NO synthase (NOS)-mediated NO production. This response varied markedly between individual patients. The MRSA-stimulated NO production was not dependent on 2 important components of bitter taste signaling: phospholipase C isoform β-2 or the transient receptor potential melastatin isoform 5 (TRPM5) ion channel. Conclusion This study shows that a S. aureus product elicits an NO-mediated innate defense response in human upper airway epithelium. The active bacterial product is likely a small, nonpeptide molecule that triggers a pathway independent of bitter taste receptors. Patient variation in the NO response to MRSA product(s), potentially due to genetic differences, might play a role in pathophysiology of Gram-positive upper respiratory infections and/or pathogenesis of chronic rhinosinusitis. PMID:26097237

  9. Demonstration of carboxylesterase in cytology samples of human nasal respiratory epithelium

    SciTech Connect

    Rodgers, D.A.; Nikula, K.J.; Avila, K.

    1995-12-01

    The epithelial lining of the nasal airways is a target for responses induced by a variety of toxicant exposures. The high metabolic capacity of this tissue has been suggested to play a role in both protection of the airways through detoxication of certain toxicants, as well as in activation of other compounds to more toxic metabolites. Specifically, nasal carboxylesterase (CE) has been shown to mediate the toxicity of inhaled esters and acrylates by converting them to more toxic acid and alcohol metabolites which can be cytotoxic and/or carcinogenic to the nasal mucosa. Due to difficulties in extrapolating rodent models to human, new paradigms using human cells and tissues are essential to understanding and evaluating the metabolic processes in human nasal epithelium.

  10. Isolation and Transplantation of Corneal Endothelial Cell–Like Cells Derived from In-Vitro-Differentiated Human Embryonic Stem Cells

    PubMed Central

    Zhang, Kai; Pang, Kunpeng

    2014-01-01

    The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell–conditioned medium. Within 1 week, CEC-like cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated cell sorting (FACS)–based isolation of the N-cadherin/vimentin dual-positive population enriches for CEC-like cells. The isolated CEC-like cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and seeded onto posterior acellular porcine corneal matrix lamellae to construct the CEC-like cell sheets. Pump function parameters of the CEC-like cell sheets approximated those of human donor corneas. Importantly, when the CEC-like cell sheets were transplanted into the eyes of rabbit CE dysfunction models, the corneal transparency was restored gradually. In conclusion, CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CEC biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future. PMID:24499373

  11. Innate immune response of human pluripotent stem cell-derived airway epithelium.

    PubMed

    McIntyre, Brendan A S; Kushwah, Rahul; Mechael, Rami; Shapovalova, Zoya; Alev, Cantas; Bhatia, Mickie

    2015-07-01

    The acquisition of innate immune response is requisite to having bona fide differentiation of airway epithelium. Procedures developed to differentiate lung airway from human pluripotent stem cells (hPSCs) have demonstrated anecdotal evidence for innate immune response, but an in-depth exploration of response levels is lacking. Herein, using an established method of airway epithelial generation from hPSCs, we show that hPSC-derived epithelial cells are able to up-regulate expression of TNFα, IL8 and IL1β in response to challenge with bacterial endotoxin LPS, but lack response from genes associated with innate immune response in other cell types. Further, stimulation of cells with TNF-α resulted in auto-induction of TNFα transcript, as well as cytokine responses of IL8 and IL1β. The demonstration of innate immune induction in hPSC-derived airway epithelia gives further strength to the functionality of in vitro protocols aimed at generating differentiated airway cells that can potentially be used in a translational setting. Finally, we propose that innate immune challenge of airway epithelium from human pluripotent stem cell sources be used as a robust validation of functional in vitro differentiation.

  12. Electric impedance of human embryonic stem cell-derived retinal pigment epithelium.

    PubMed

    Onnela, Niina; Savolainen, Virpi; Juuti-Uusitalo, Kati; Vaajasaari, Hanna; Skottman, Heli; Hyttinen, Jari

    2012-02-01

    The barrier properties of epithelium are conventionally defined by transepithelial resistance (TER). TER provides information about the tightness of the epithelium. Electrical impedance spectroscopy (EIS) provides additional information regarding cell membrane properties, such as changes in electric capacitance and possible parallel or serial pathways that may correlate with the morphology of the cell layer. This study presents EIS of retinal pigment epithelial (RPE) cell model of the putative RPE differentiated from human embryonic stem cells (hESC-RPE). The generally utilized RPE cell model, ARPE-19, was used as immature control. The measured EIS was analyzed by fitting an equivalent electrical circuit model describing the resistive and capacitive properties of the RPE. Our results indicated that TER of hESC-RPE cells was close to the values of human RPE presented in the literature. This provides evidence that the stem cell-derived RPE in vitro can reach high-barrier function. Furthermore, hESC-RPE cells produced impedance spectra that can be modeled by the equivalent circuit of one time constant. ARPE-19 cells produced low-barrier properties, that is, an impedance spectra that suggested poor maturation of ARPE-19 cells. To conclude, EIS could give us means for non-invasively estimating the functionality and maturation of differentiated-RPE cells.

  13. Circular flow patterns induced by ciliary activity in reconstituted human bronchial epithelium

    NASA Astrophysics Data System (ADS)

    Viallat, Annie; Khelloufi, Kamel; Gras, Delphine; Chanez, Pascal; Aix Marseille Univ., CNRS, CINaM, Marseille, France Team; Aix Marseille Univ., CNRS, Inserm, LAI, Marseille, France Team

    2016-11-01

    Mucociliary clearance is the transport at the surface of airways of a complex fluid layer, the mucus, moved by the beats of microscopic cilia present on epithelial ciliated cells. We explored the coupling between the spatial organisation and the activity of cilia and the transport of surface fluids on reconstituted cultures of human bronchial epithelium at air-liquid interface, obtained by human biopsies. We reveal the existence of stable local circular surface flow patterns of mucus or Newtonian fluid at the epithelium surface. We find a power law over more than 3 orders of magnitude showing that the average ciliated cell density controls the size of these flow patterns, and, therefore the distance over which mucus can be transported. We show that these circular flow patterns result from the radial linear increase of the local propelling forces (due to ciliary beats) on each flow domain. This linear increase of local forces is induced by a fine self-regulation of both cilia density and orientation of ciliary beats. Local flow domains grow and merge during ciliogenesis to provide macroscopic mucus transport. This is possible only when the viscoelastic mucus continuously exerts a shear stress on beating cilia, revealing a mechanosensitive function of cilia. M. K. Khelloufi thanks the society MedBioMed for financial support. This work was supported by the ANR MUCOCIL project, Grant ANR-13-BSV5-0015 of the French Agence Nationale de la Recherche.

  14. A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells

    PubMed Central

    Ogaki, Soichiro; Morooka, Mayu; Otera, Kaito; Kume, Shoen

    2015-01-01

    The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes. PMID:26616277

  15. The chronicles of Porphyromonas gingivalis: the microbium, the human oral epithelium and their interplay.

    PubMed

    Yilmaz, Ozlem

    2008-10-01

    The microbiota of the human oral mucosa consists of a myriad of bacterial species that normally exist in commensal harmony with the host. Porphyromonas gingivalis, an aetiological agent in severe forms of periodontitis (a chronic inflammatory disease), is a prominent component of the oral microbiome and a successful colonizer of the oral epithelium. This Gram-negative anaerobe can also exist within the host epithelium without the existence of overt disease. Gingival epithelial cells, the outer lining of the gingival mucosa, which function as an important part of the innate immune system, are among the first host cells colonized by P. gingivalis. This review describes recent studies implicating the co-existence and intracellular adaptation of the organism in these target host cells. Specifically, recent findings on the putative mechanisms of persistence, intercellular dissemination and opportunism are highlighted. These new findings may also represent an original and valuable model for mechanistic characterization of other successful host-adapted, self-limiting, persistent intracellular bacteria in human epithelial tissues.

  16. HIV-associated disruption of mucosal epithelium facilitates paracellular penetration by human papillomavirus.

    PubMed

    Tugizov, Sharof M; Herrera, Rossana; Chin-Hong, Peter; Veluppillai, Piri; Greenspan, Deborah; Michael Berry, J; Pilcher, Christopher D; Shiboski, Caroline H; Jay, Naomi; Rubin, Mary; Chein, Aung; Palefsky, Joel M

    2013-11-01

    The incidence of human papillomavirus (HPV)-associated epithelial lesions is substantially higher in human immunodeficiency virus (HIV)-infected individuals than in HIV-uninfected individuals. The molecular mechanisms underlying the increased risk of HPV infection in HIV-infected individuals are poorly understood. We found that HIV proteins tat and gp120 were expressed within the oral and anal mucosal epithelial microenvironment of HIV-infected individuals. Expression of HIV proteins in the mucosal epithelium was correlated with the disruption of epithelial tight junctions (TJ). Treatment of polarized oral, cervical and anal epithelial cells, and oral tissue explants with tat and gp120 led to disruption of epithelial TJ and increased HPV pseudovirion (PsV) paracellular penetration in to the epithelium. PsV entry was observed in the basal/parabasal cells, the cells in which the HPV life cycle is initiated. Our data suggest that HIV-associated TJ disruption of mucosal epithelia may potentiate HPV infection and subsequent development of HPV-associated neoplasia.

  17. Coculture of dorsal root ganglion neurons and differentiated human corneal stromal stem cells on silk-based scaffolds.

    PubMed

    Wang, Siran; Ghezzi, Chiara E; White, James D; Kaplan, David L

    2015-10-01

    Corneal tissue displays the highest peripheral nerve density in the human body. Engineering of biomaterials to promote interactions between neurons and corneal tissue could provide tissue models for nerve/cornea development, platforms for drug screening, as well as innovative opportunities to regenerate cornea tissue. The focus of this study was to develop a coculture system for differentiated human corneal stromal stem cells (dhCSSCs) and dorsal root ganglion neurons (DRG) to mimic the human cornea tissue interactions. Axon extension, connectivity, and neuron cell viability were studied. DRG neurons developed longer axons when cocultured with dhCSSCs in comparison to neuron cultures alone. To assess the mechanism involved in the coculture response, nerve growth factors (NGF) secreted by dhCSSCs including NGF, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and neurotrophin-3 were characterized with greater focus on BDNF secretion. DhCSSCs also secreted collagen type I, an extracellular matrix molecule favorable for neuronal outgrowth. This coculture system provides a slowly degrading silk matrix to study neuronal responses in concert with hCSSCs related to innervation of corneal tissue with utility toward human corneal nerve regeneration and associated diseases. © 2015 Wiley Periodicals, Inc.

  18. In vivo three-dimensional imaging of human corneal nerves using Fourier-domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Shin, Jun Geun; Hwang, Ho Sik; Eom, Tae Joong; Lee, Byeong Ha

    2017-01-01

    We have employed Fourier-domain optical coherence tomography (FD-OCT) to achieve corneal nerve imaging, which could be useful in surgical planning and refractive surgery. Because the three-dimensional (3-D) images of the corneal nerves were acquired in vivo, unintentional movement of the subject during the measurement led to imaging artifacts. These artifacts were compensated for with a series of signal processing techniques, namely realigning A-scan images to flatten the boundary and cross-correlating adjacent B-scan images. To overcome the undesirably large signal from scattering at the corneal surface and iris, volume rendering and maximum intensity projections were performed with only the data taken in the stromal region of the cornea, which is located between 200 and 500 μm from the corneal surface. The 3-D volume imaging of a 10×10 mm2 area took 9.8 s, which is slightly shorter than the normal tear breakup time. This allowed us to image the branched and threadlike corneal nerve bundles within the human eye. The experimental results show that FD-OCT systems have the potential to be useful in clinical investigations of corneal nerves and by minimizing nerve injury during clinical or surgical procedures.

  19. A novel quantitative methodology for age evaluation of the human corneal endothelium

    NASA Astrophysics Data System (ADS)

    Rannou, Klervi; Thuret, Gilles; Gain, Philippe; Pinoli, Jean-Charles; Gavet, Yann

    2017-03-01

    The human corneal endothelium regulates the cornea transparency. Its cells, that cannot regenerate after birth, form a tesselated mosaic with almost perfect hexagonal cells during childhood, becoming progressively bigger and less ordered during aging. This study included 50 patients (in 10 decades groups) and 10 specular microscopy observations per patient. Five different criteria were measured on the manually segmented cells: area and perimeter of the cells as well as reduced Minkowski functionals. All these criteria were used to assess the probability of age group membership. We demonstrated that the age evaluation is near the reality, although a high variability was observed for patients between 30 and 70 years old.

  20. [The research advance of small molecules inducing embryonic stem cells to differentiate into corneal epithelial-like cells].

    PubMed

    Qin, L M; Chen, H; Huang, Y F

    2017-03-11

    Stem cells are becoming a hot topic of basic medicine and clinical research because of their wide self-renewal and differentiation potential in recent years. A number of small molecules that can be used to control stem cell self-renewal, lineage differentiation, reprogramming and regeneration could regulate stem cell fate quickly and reversibly. If small molecules could induce human embryonic stem cells to differentiate into corneal epithelial-like cells in vivo, corneal cell transplantation therapy will have unlimited source of corneal epithelium. More patients will obtain effective and timely treatment. (Chin J Ophthalmol, 2017, 53: 230-235).

  1. Corneal Disorders

    MedlinePlus

    ... Injuries Dystrophies - conditions in which parts of the cornea lose clarity due to a buildup of cloudy material Treatments of corneal disorders include medicines, corneal transplantation, and corneal laser surgery. NIH: National Eye Institute

  2. Quality control in microarray assessment of gene expression in human airway epithelium

    PubMed Central

    Raman, Tina; O'Connor, Timothy P; Hackett, Neil R; Wang, Wei; Harvey, Ben-Gary; Attiyeh, Marc A; Dang, David T; Teater, Matthew; Crystal, Ronald G

    2009-01-01

    Background Microarray technology provides a powerful tool for defining gene expression profiles of airway epithelium that lend insight into the pathogenesis of human airway disorders. The focus of this study was to establish rigorous quality control parameters to ensure that microarray assessment of the airway epithelium is not confounded by experimental artifact. Samples (total n = 223) of trachea, large and small airway epithelium were collected by fiberoptic bronchoscopy of 144 individuals and hybridized to Affymetrix microarrays. The pre- and post-chip quality control (QC) criteria established, included: (1) RNA quality, assessed by RNA Integrity Number (RIN) ≥ 7.0; (2) cRNA transcript integrity, assessed by signal intensity ratio of GAPDH 3' to 5' probe sets ≤ 3.0; and (3) the multi-chip normalization scaling factor ≤ 10.0. Results Of the 223 samples, all three criteria were assessed in 191; of these 184 (96.3%) passed all three criteria. For the remaining 32 samples, the RIN was not available, and only the other two criteria were used; of these 29 (90.6%) passed these two criteria. Correlation coefficients for pairwise comparisons of expression levels for 100 maintenance genes in which at least one array failed the QC criteria (average Pearson r = 0.90 ± 0.04) were significantly lower (p < 0.0001) than correlation coefficients for pairwise comparisons between arrays that passed the QC criteria (average Pearson r = 0.97 ± 0.01). Inter-array variability was significantly decreased (p < 0.0001) among samples passing the QC criteria compared with samples failing the QC criteria. Conclusion Based on the aberrant maintenance gene data generated from samples failing the established QC criteria, we propose that the QC criteria outlined in this study can accurately distinguish high quality from low quality data, and can be used to delete poor quality microarray samples before proceeding to higher-order biological analyses and interpretation. PMID:19852842

  3. Morphological Characterization of Organized Extracellular Matrix Deposition by Ascorbic Acid-Stimulated Human Corneal Fibroblasts

    PubMed Central

    Guo, Xiaoqing; Hutcheon, Audrey E. K.; Melotti, Suzanna A.; Zieske, James D.; Trinkaus-Randall, Vickery; Ruberti, Jeffrey W.

    2016-01-01

    Purpose To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures. Methods Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of Vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to five weeks. Constructs were assessed via light (phase contrast and differential interference contrast) and transmission (standard and quick freeze/deep etch) microscopy. Results Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27–51 nm) which often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct while quick-freeze deep etch micrographs showed the details of the matrix interaction with fibroblasts via arrays of membrane surface structures. Conclusions Human keratocytes, cultured in a stable Vitamin C derivative, are capable of assembling extracellular matrix which comprise parallel arrays of ECM fibrils. The resulting constructs, which are highly cellular, exhibit morphology similar to the developing mammalian stroma where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggest a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes which govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue-engineering a biomimetic stroma. PMID:17724187

  4. Corneal Endothelial Cell Integrity in Precut Human Donor Corneas Enhanced by Autocrine Vasoactive Intestinal Peptide

    PubMed Central

    Coll, Timothy; Gloria, Dante; Sprehe, Nicholas

    2017-01-01

    Purpose: To demonstrate that vasoactive intestinal peptide (VIP), a corneal endothelial (CE) cell autocrine factor, maintains the integrity of corneal endothelium in human donor corneoscleral explants precut for endothelial keratoplasty. Methods: Twelve paired human donor corneoscleral explants used as control versus VIP-treated explants (10 nM, 30 minutes, 37°C) were shipped (4°C) to the Lions Eye Institute for Transplantation and Research for precutting (Moria CBM-ALTK Keratome), shipped back to the laboratory, and cultured in ciliary neurotrophic factor (CNTF, 0.83 nM, 37°C, 24 hours). Trephined endothelial discs (8–8.5 mm) were analyzed for differentiation markers (N-cadherin, CNTF receptor α subunit [CNTFRα], and connexin 43) by Western blot after a quarter of the discs from 4 paired explants were cut away and stained with alizarin red S for microscopic damage analysis. Two additional paired explants (6 days in culture) were stained for panoramic view of central CE damage. Results: VIP treatment increased N-cadherin and CNTFRα levels (mean ± SEM) to 1.38 ± 0.11-fold (P = 0.003) and 1.46 ± 0.22-fold (P = 0.03) of paired controls, respectively, whereas CE cell CNTF responsiveness in upregulation of connexin 43 increased to 2.02 ± 0.5 (mean ± SEM)-fold of the controls (P = 0.04). CE damage decreased from (mean ± SEM) 10.0% ± 1.2% to 1.6% ± 0.3% (P < 0.0001) and 9.1% ± 1.1% to 2.4% ± 1.0% (P = 0.0006). After 6 days in culture, the damage in whole CE discs decreased from 20.0% (control) to 5.5% (VIP treated). Conclusions: VIP treatment before precut enhanced the preservation of corneal endothelium. PMID:28181929

  5. High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells

    PubMed Central

    Ye, J; Wu, H; Wu, Y; Wang, C; Zhang, H; Shi, X; Yang, J

    2012-01-01

    Purpose To investigate the toxic effects of ethylenediaminetetraacetic acid disodium salt (EDTA), a corneal penetration enhancer in topical ophthalmic formulations, on DNA in human corneal epithelial cells (HCEs), and to investigate whether the effect induced by EDTA can be inhibited by high molecular weight hyaluronan (HA). Methods Cells were exposed to EDTA in concentrations ranging from 0.00001 to 0.01% for 60 min, or 30 min high molecular weight HA pretreatment followed by EDTA treatment. The cell viability was measured by the MTT test. Cell apoptosis was determined with annexin V staining by flow cytometry. The DNA single- and double-strand breaks of HCEs were examined by alkaline comet assay and by immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci, respectively. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2′, 7′-dichlorodihydrofluorescein diacetate. Results EDTA exhibited no adverse effect on cell viability and did not induce cell apoptosis in human corneal epithelial cells at concentrations lower than 0.01%. However, a significant increase of DNA single- and double-strand breaks was observed in a dose-dependent manner with all the concentrations of EDTA tested in HCEs. In addition, EDTA treatment led to elevated ROS generation. Moreover, 30 min preincubation with high molecular weight HA significantly decreased EDTA-induced ROS generation and DNA damage. Conclusions EDTA could induce DNA damage in HCEs, probably through oxidative stress. Furthermore, high molecular weight HA was an effective protective agent that had antioxidant properties and decreased DNA damage induced by EDTA. PMID:22595911

  6. Laminin-511 and -521 enable efficient in vitro expansion of human corneal endothelial cells.

    PubMed

    Okumura, Naoki; Kakutani, Kazuya; Numata, Ryohei; Nakahara, Makiko; Schlötzer-Schrehardt, Ursula; Kruse, Friedrich; Kinoshita, Shigeru; Koizumi, Noriko

    2015-05-01

    The purpose of this study was to investigate the usefulness of laminin isoforms as substrates for culturing human corneal endothelial cells (HCECs) for clinical application of tissue engineering therapy. Expression of specific laminin chains in human corneal endothelium and Descemet's membrane was analyzed at the mRNA and protein levels. The effect of laminin-511 and -521 on cell adhesion and proliferation was evaluated. Recombinant laminin E8 fragments (E8s), which represent functionally minimal forms of laminins, were also evaluated for their effects on cell density and cellular phenotype. The potential involvement of α3β1 and α6β1 integrins in laminin signal transduction was also investigated using neutralizing antibodies. Laminin-511 and -521 were expressed in Descemet's membrane and corneal endothelium. These laminin isoforms significantly enhanced the in vitro adhesion and proliferation, and differentiation of HCECs. A cell density of 2200 to 2400 cells/mm2 was achieved when HCECs were cultured on laminin-511 or -521, whereas the density was only 1100 cells/mm2 on an uncoated control. E8s also supported HCEC cultivation with a similar efficacy to that obtained with full-length laminin. Functional blocking of α3β1 and α6β1 integrins suppressed the adhesion of HCECs even in the presence of laminin-511. Laminin-511 and -521 were the laminin isoforms present in Descemet's membrane, and these laminins modulate the adhesion and proliferation of CECs. Laminin E8s represent an ideal xeno-free defined substrate for the culture of CECs for clinical applications.

  7. Vitamin D Activation and Function in Human Corneal Epithelial Cells During TLR-Induced Inflammation

    PubMed Central

    Reins, Rose Yvonne; Baidouri, Hasna; McDermott, Alison Marie

    2015-01-01

    Purpose Vitamin D is recognized to be an important modulator of the immune system. In the eye, studies have shown that deficiencies and genetic differences in vitamin D–related genes have a significant impact on the development of various ocular diseases. Our current study examines the ability of human corneal epithelial cells (HCEC) to activate vitamin D and the effect of vitamin D treatment on antimicrobial peptide production and cytokine modulation during inflammation, with the ultimate goal of using vitamin D therapeutically for corneal inflammation. Methods Human corneal epithelial cells were treated with 10−7M vitamin D3 (D3) or 25-hydroxyvitamin D3 (25D3) for 24 hours and 1,25-dihydroxyvitamin D3 (1,25D3) detected by immunoassay. Human cathelicidin (LL-37) expression was examined by RT-PCR, immunoblot, and immunostaining following 1,25D3 treatment and antimicrobial activity of 1,25D3-treated cells was determined. Cells were stimulated with TLR3 agonist polyinosinic-polycytidylic acid (Poly[I:C]) for 24 hours and cytokine levels measured by RT-PCR, ELISA, and Luminex. Immunostaining determined expression of vitamin D receptor (VDR) and retinoic acid inducible gene-1 receptor (RIG-1) as well as NF-κB nuclear translocation. Results When treated with inactive vitamin D metabolites, HCEC produced active 1,25D3, leading to enhanced expression of the antimicrobial peptide, LL-37, dependent on VDR. 1,25-D3 decreased the expression of proinflammatory cytokines (IL-1β, IL-6, TNFα, and CCL20) and MMP-9 induced by Poly(I:C) as well as pattern recognition receptor expression (TLR3, RIG-1, MDA5). However, early activation of NF-κB was not affected. Conclusions These studies demonstrate the protective ability of vitamin D to attenuate proinflammatory mediators while increasing antimicrobial peptides and antipseudomonas activity in corneal cells, and further our knowledge on the immunomodulatory functions of the hormone. PMID:26641549

  8. Does the adult human ciliary body epithelium contain "true" retinal stem cells?

    PubMed

    Frøen, Rebecca; Johnsen, Erik O; Nicolaissen, Bjørn; Facskó, Andrea; Petrovski, Goran; Moe, Morten C

    2013-01-01

    Recent reports of retinal stem cells being present in several locations of the adult eye have sparked great hopes that they may be used to treat the millions of people worldwide who suffer from blindness as a result of retinal disease or injury. A population of proliferative cells derived from the ciliary body epithelium (CE) has been considered one of the prime stem cell candidates, and as such they have received much attention in recent years. However, the true nature of these cells in the adult human eye has still not been fully elucidated, and the stem cell claim has become increasingly controversial in light of new and conflicting reports. In this paper, we will try to answer the question of whether the available evidence is strong enough for the research community to conclude that the adult human CE indeed harbors stem cells.

  9. [Decellularized collagen matrix from tilapia fish scales for corneal reconstruction (BioCornea)].

    PubMed

    Hos, D; van Essen, T H; Bock, F; Chou, C-H; Pan, H-A; Lin, C-C; Huang, M-C; Chen, S-C; Cursiefen, C; Jager, M J

    2014-11-01

    The worldwide need for donor corneal tissue clearly exceeds the availability of transplantable human tissue; therefore, recent efforts aim to identify and characterize alternative tissues, such as decellularized collagen scaffolds. The transparent fish scales of tilapia (Oreochromis mossambicus) were analyzed as a potential alternative for corneal reconstruction ("BioCornea"). The article gives a review of the literature and own preliminary results. After decellularization the tissue characteristics of the fish scales, the repopulation with corneal epithelium and stromal cells, immunogenicity, the feasibility of corneal transplantation and the angiogenic properties were analyzed in vitro and in various animal models. The fish scales mainly consist of collagen type I and show an architecture that is similar to the human cornea. Corneal epithelium and stromal cells are able to grow over and into the scaffold. It is possible to transplant fish scales in various animal models without severe inflammatory responses. Furthermore, in mice, less blood and lymphatic vessels grow into the xenograft when compared to conventional allogenic transplants. Preliminary results with decellularized tilapia fish scales as an alternative for corneal reconstruction ("BioCornea") are promising.

  10. Corneal haze induced by excimer laser photoablation in rabbits is reduced by preserved human amniotic membrane graft

    NASA Astrophysics Data System (ADS)

    Wang, Ming X.; Gray, Trevor; Prabhasawat, Pinnita; Ma, Xiong; Culbertson, William; Forster, Richard; Hanna, Khalil; Tseng, Scheffer C. G.

    1998-06-01

    We conducted a study to determine if preserved human amniotic membrane can reduce corneal haze induced by excimer laser photoablation. Excimer photoablation was performed bilaterally on 40 New Zealand white rabbits with a 6 mm ablation zone and 120 micrometer depth (PTK) using the VISX Star. One eye was randomly covered with a preserved human amniotic membrane and secured using four interrupted 10 - 0 nylon sutures; the other eye served as control. The amniotic membranes were removed at one week, and the corneal haze was graded with a slit-lamp biomicroscopy by three masked corneal specialists (WC, KH and RF) biweekly for the ensuing 12 weeks. Histology and in situ TUNEL staining (for fragmented DNA as an index for apoptosis) was performed at days 1, 3 and 7 and at 12 weeks. One week after excimer photoablation, the amniotic membrane-covered corneas showed more anterior stromal edema, which resolved at the second week. A consistent grading of organized reticular corneal haze was noted among the three masked observers. Such corneal haze peaked at the seventh week in both groups. The amniotic membrane-covered group showed statistically significant less corneal haze (0.50 plus or minus 0.15) than the control groups (1.25 plus or minus 0.35) (p less than 0.001). The amniotic membrane-covered corneas had less inflammatory response at days 1 and 3, showing nearly nil DNA fragmentation on keratocytes on the ablated anterior stromal and less stromal fibroblast activation. There is less altered epithelial cell morphology and less epithelial hyperplasia at 1 week in these amniotic membrane-treated eyes. We concluded from this study that amniotic membrane matrix is effective in reducing corneal haze induced by excimer photoablation in rabbits and may have clinical applications.

  11. FOXJ1 prevents cilia growth inhibition by cigarette smoke in human airway epithelium in vitro.

    PubMed

    Brekman, Angelika; Walters, Matthew S; Tilley, Ann E; Crystal, Ronald G

    2014-11-01

    Airway epithelium ciliated cells play a central role in clearing the lung of inhaled pathogens and xenobiotics, and cilia length and coordinated beating are important for airway clearance. Based on in vivo studies showing that the airway epithelium of healthy smokers has shorter cilia than that of healthy nonsmokers, we investigated the mechanisms involved in cigarette smoke-mediated inhibition of ciliogenesis by assessing normal human airway basal cell differentiation in air-liquid interface (ALI) cultures in the presence of nontoxic concentrations of cigarette smoke extract (CSE). Measurements of cilia length from Day 28 ALI cultures demonstrated that CSE exposure was associated with shorter cilia (P < 0.05), reproducing the effect of cigarette smoking on cilia length observed in vivo. This phenotype correlated with a broad CSE-mediated suppression of genes involved in cilia-related transcriptional regulation, intraflagellar transport, cilia motility, structural integrity, and basal body development but not of control genes or epithelial barrier integrity. The CSE-mediated inhibition of cilia growth could be prevented by lentivirus-mediated overexpression of FOXJ1, the major cilia-related transcription factor, which led to partial reversal of expression of cilia-related genes suppressed by CSE. Together, the data suggest that components of cigarette smoke are responsible for a broad suppression of genes involved in cilia growth, but, by stimulating ciliogenesis with the transcription factor FOXJ1, it may be possible to maintain close to normal cilia length despite the stress of cigarette smoking.

  12. Differential response of the epithelium and interstitium in developing human fetal lung explants to hyperoxia.

    PubMed

    Bustani, Porus; Hodge, Rachel; Tellabati, Ananth; Li, Juan; Pandya, Hitesh; Kotecha, Sailesh

    2006-03-01

    Hyperoxia is closely linked with the development of chronic lung disease of prematurity (CLD), but the exact mechanisms whereby hyperoxia alters the lung architecture in the developing lung remain largely unknown. We developed a fetal human lung organ culture model to investigate (a) the morphologic changes induced by hyperoxia and (b) whether hyperoxia resulted in differential cellular responses in the epithelium and interstitium. The effects of hyperoxia on lung morphometry were analyzed using computer-assisted image analysis. The lung architecture remained largely unchanged in normoxia lasting as long as 4 d. In contrast, hyperoxic culture of pseudoglandular fetal lungs resulted in significant dilatation of airways, thinning of the epithelium, and regression of the interstitium including the pulmonary vasculature. Although there were no significant differences in Ki67 between normoxic and hyperoxic lungs, activated caspase-3 was significantly increased in interstitial cells, but not epithelial cells, under hyperoxic conditions. These changes show that exposure of pseudoglandular lungs to hyperoxia modulates the lung architecture to resemble saccular lungs.

  13. TNFα Affects Ciliary Beat Response to Increased Viscosity in Human Pediatric Airway Epithelium.

    PubMed

    González, Claudia; Droguett, Karla; Rios, Mariana; Cohen, Noam A; Villalón, Manuel

    2016-01-01

    In airway epithelium, mucociliary clearance (MCC) velocity depends on the ciliary beat frequency (CBF), and it is affected by mucus viscoelastic properties. Local inflammation induces secretion of cytokines (TNFα) that can alter mucus viscosity; however airway ciliated cells have an autoregulatory mechanism to prevent the collapse of CBF in response to increase in mucus viscosity, mechanism that is associated with an increment in intracellular Ca(+2) level ([Ca(2+)]i). We studied the effect of TNFα on the autoregulatory mechanism that regulates CBF in response to increased viscosity using dextran solutions, in ciliated cells cultured from human pediatric epithelial adenoid tissue. Cultures were treated with TNFα, before and after the viscous load was changed. TNFα treatment produced a significantly larger decrease in CBF in cultures exposed to dextran. Furthermore, an increment in [Ca(2+)]i was observed, which was significantly larger after TNFα treatment. In conclusion, although TNFα has deleterious effects on ciliated cells in response to maintaining CBF after increasing viscous loading, it has a positive effect, since increasing [Ca(2+)]i may prevent the MCC collapse. These findings suggest that augmented levels of TNFα associated with an inflammatory response of the nasopharyngeal epithelium may have dual effects that contribute to maintaining the effectiveness of MCC in the upper airways.

  14. TNFα Affects Ciliary Beat Response to Increased Viscosity in Human Pediatric Airway Epithelium

    PubMed Central

    Droguett, Karla; Rios, Mariana; Cohen, Noam A.

    2016-01-01

    In airway epithelium, mucociliary clearance (MCC) velocity depends on the ciliary beat frequency (CBF), and it is affected by mucus viscoelastic properties. Local inflammation induces secretion of cytokines (TNFα) that can alter mucus viscosity; however airway ciliated cells have an autoregulatory mechanism to prevent the collapse of CBF in response to increase in mucus viscosity, mechanism that is associated with an increment in intracellular Ca+2 level ([Ca2+]i). We studied the effect of TNFα on the autoregulatory mechanism that regulates CBF in response to increased viscosity using dextran solutions, in ciliated cells cultured from human pediatric epithelial adenoid tissue. Cultures were treated with TNFα, before and after the viscous load was changed. TNFα treatment produced a significantly larger decrease in CBF in cultures exposed to dextran. Furthermore, an increment in [Ca2+]i was observed, which was significantly larger after TNFα treatment. In conclusion, although TNFα has deleterious effects on ciliated cells in response to maintaining CBF after increasing viscous loading, it has a positive effect, since increasing [Ca2+]i may prevent the MCC collapse. These findings suggest that augmented levels of TNFα associated with an inflammatory response of the nasopharyngeal epithelium may have dual effects that contribute to maintaining the effectiveness of MCC in the upper airways. PMID:28025644

  15. Corneal endothelial autocrine trophic factor VIP in a mechanism-based strategy to enhance human donor cornea preservation for transplantation.

    PubMed

    Koh, Shay-Whey Margaret

    2012-02-01

    Vasoactive intestinal peptide (VIP) and ciliary neurotrophic factor (CNTF) are identified as autocrines of human corneal endothelial (CE) cells working in concert to maintain the differentiated state and promote the survival of the corneal endothelium. From VIP gene knockdown study, endogenous VIP is shown to maintain the level of the differentiation marker, the adhesion molecule N-cadherin, CE cell size, shape, and retention, in situ in the human donor corneoscleral explants. Exogenous VIP protects the corneal endothelium against the killing effect of oxidative stress, in part by upholding ATP levels in CE cells dying of oxidative stress-induced injury, allowing them to die of an apoptotic death instead of an acute necrotic one. The switch from the acute necrosis to the programmed cell death (apoptosis) may have allowed the injured CE cell to be rescued by the VIP-upregulated pathways, including those of Bcl-2 and N-cadherin, and resulted in long-term CE cell survival. The endogenous VIP in CE cells is upregulated by CNTF, which is released by CE cells surviving the oxidative stress. The CNTF receptor (CNTFRα) is expressed in CE cells in human donor corneoscleral explant and gradually becomes lost during corneal storage. VIP treatment (10(-8) M, 37 °C, 30 min) prior to storage of freshly dissected human donor corneoscleral explants increases their CE cell CNTFRα level and responsiveness to CNTF in upregulating the gap junctional protein connexin-43 expression. VIP treatment of both fresh and preserved corneoscleral explants reduces CE damage in the corneoscleral explants and in the corneal buttons trephined from them. CE cell loss is a critical risk factor in corneal graft failure at any time in the life of the graft, which can be as late as 5-10 years after an initially successful transplant. A new procedure, Descemet's stripping automated endothelial keratoplasty (DSAEK), which is superior to the traditional full thickness transplantation in many aspects

  16. Effect of cysteamine on oxidative stress-induced cell death of human corneal endothelial cells.

    PubMed

    Shin, Young Joo; Seo, Jong Mo; Chung, Tae Young; Hyon, Joon Young; Wee, Won Ryang

    2011-10-01

    The principal objective of this study was to evaluate the protective effect of cysteamine against the oxidative stress-induced cell death of human corneal endothelial cells. In this study, human corneal endothelial cells (HCECs) were cultured according to a previously published method. With treatment of 0 mM or 5 mM of tert-butyl hydroperoxide (tBHP) with various concentrations (0-50 mM) of cysteamine, reactive oxygen species (ROS) production was measured using an oxidation-sensitive fluorescent probe, and dichlorofluorescein diacetate (DCFH-DA) methods. Cell viability was assayed via the Cell Counting Kit-8 method. The levels of cellular glutathione were also assessed enzymatically with glutathione reductase using a commercial glutathione assay kit (Cayman Chemical, USA). This study showed that cysteamine reduced 2',7'-dihydrodichlorofluorescein oxidation and increased glutathione. Cysteamine significantly inhibited tBHP-induced ROS production. Cysteamine-treated cells evidenced higher viability relative to the controls at 5 mM tBHP, and cysteamine also effectively protected HCECs against ROS-induced cell death via an increase in intracellular glutathione. Our data indicate that cysteamine was not toxic at low concentrations and, at high concentrations, protects HCECs against oxidative injury-mediated cell death via the inhibition of ROS production, although cysteamine is toxic in cells at high concentrations without oxidative stress.

  17. Combined chlorhexidine and PVP-I decontamination of human donor eyes prior to corneal preservation.

    PubMed

    van Luijk, Chantal M; Bruinsma, Marieke; van der Wees, Jacqueline; Lie, Jessica T; Ham, Lisanne; Melles, Gerrit R J

    2012-06-01

    The aim of this study was to report the efficacy of adding chlorhexidine to the protocol for decontamination of human donor globes prior to excision of corneo-scleral rims for future keratoplasty procedures. In 2005, chlorhexidine was introduced by our eye bank as an additional step in the protocol for decontaminating human donor globes. After 5 years, we prospectively evaluated the number of contaminations. Out of 2,891 globes included in our study, 2,663 globes were processed, of which 36 (1.4%) were considered contaminated. Seventeen contaminations (0.6%) were detected by culturing limbal swabs, directly after decontamination, eight (0.3%) by visible discoloration of the culture medium carrying a corneo-scleral rim, and eleven (0.4%) after inoculation of the culture medium on blood agar plates. Importantly, after 4 weeks of incubation, none of the aerobic and anaerobic cultures taken from the secondary 'transport medium' (dextran containing medium used to transport corneal tissue to the transplantation centre) showed microbiological growth. In conclusion, the combined use of 0.02% chlorhexidine and 0.5% povidone-iodine may allow decontamination of donor globes to a level at which the risk of tissue contamination at the time of transplantation is minimized, while corneal viability is preserved.

  18. Generation of novel AAV variants by directed evolution for improved CFTR delivery to human ciliated airway epithelium.

    PubMed

    Li, Wuping; Zhang, Liqun; Johnson, Jarrod S; Zhijian, Wu; Grieger, Joshua C; Ping-Jie, Xiao; Drouin, Lauren M; Agbandje-McKenna, Mavis; Pickles, Raymond J; Samulski, R Jude

    2009-12-01

    Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.

  19. Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

    PubMed Central

    Li, Wuping; Zhang, Liqun; Johnson, Jarrod S; Zhijian, Wu; Grieger, Joshua C; Ping-Jie, Xiao; Drouin, Lauren M; Agbandje-McKenna, Mavis; Pickles, Raymond J; Samulski, R Jude

    2009-01-01

    Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease. PMID:19603002

  20. Claudin-19 and the Barrier Properties of the Human Retinal Pigment Epithelium

    PubMed Central

    Peng, Shaomin; Rao, Veena S.; Adelman, Ron A.

    2011-01-01

    Purpose. The retinal pigment epithelium (RPE) separates photoreceptors from choroidal capillaries, but in age-related macular degeneration (AMD) capillaries breach the RPE barrier. Little is known about human RPE tight junctions or the effects of serum on the retinal side of the RPE. Methods. Cultured human fetal RPE (hfRPE) was assessed by the transepithelial electrical resistance (TER) and the transepithelial diffusion of methylated polyethylene glycol (mPEG). Claudins and occludin were monitored by quantitative RT-PCR, immunoblotting, and immunofluorescence. Results. Similar to freshly isolated hfRPE, claudin-19 mRNA was 25 times more abundant than claudin-3. Other detectable claudin mRNAs were found in even lesser amounts, as little as 3000 times less abundant than claudin-19. Claudin-1 and claudin-10b were detected only in subpopulations of cells, whereas others were undetectable. Knockdown of claudin-19 by small interfering RNA (siRNA) eliminated the TER. siRNAs for other claudins had minimal effects. Serum affected tight junctions only when presented to the retinal side of the RPE. The TER increased 2 times, and the conductance of K+ relative to Na+ decreased without affecting the permeability of mPEG. These effects correlated with increased steady-state levels of occludin. Conclusions. Fetal human RPE is a claudin-19–dominant epithelium that has regional variations in claudin-expression. Apical serum decreases RPE permeability, which might be a defense mechanism that would retard the spread of edema due to AMD. PMID:21071746

  1. Gene Expression and Functional Annotation of the Human and Mouse Choroid Plexus Epithelium

    PubMed Central

    Janssen, Sarah F.; van der Spek, Sophie J. F.; ten Brink, Jacoline B.; Essing, Anke H. W.; Gorgels, Theo G. M. F.; van der Spek, Peter J.; Jansonius, Nomdo M.; Bergen, Arthur A. B.

    2013-01-01

    Background The choroid plexus epithelium (CPE) is a lobed neuro-epithelial structure that forms the outer blood-brain barrier. The CPE protrudes into the brain ventricles and produces the cerebrospinal fluid (CSF), which is crucial for brain homeostasis. Malfunction of the CPE is possibly implicated in disorders like Alzheimer disease, hydrocephalus or glaucoma. To study human genetic diseases and potential new therapies, mouse models are widely used. This requires a detailed knowledge of similarities and differences in gene expression and functional annotation between the species. The aim of this study is to analyze and compare gene expression and functional annotation of healthy human and mouse CPE. Methods We performed 44k Agilent microarray hybridizations with RNA derived from laser dissected healthy human and mouse CPE cells. We functionally annotated and compared the gene expression data of human and mouse CPE using the knowledge database Ingenuity. We searched for common and species specific gene expression patterns and function between human and mouse CPE. We also made a comparison with previously published CPE human and mouse gene expression data. Results Overall, the human and mouse CPE transcriptomes are very similar. Their major functionalities included epithelial junctions, transport, energy production, neuro-endocrine signaling, as well as immunological, neurological and hematological functions and disorders. The mouse CPE presented two additional functions not found in the human CPE: carbohydrate metabolism and a more extensive list of (neural) developmental functions. We found three genes specifically expressed in the mouse CPE compared to human CPE, being ACE, PON1 and TRIM3 and no human specifically expressed CPE genes compared to mouse CPE. Conclusion Human and mouse CPE transcriptomes are very similar, and display many common functionalities. Nonetheless, we also identified a few genes and pathways which suggest that the CPE between mouse and

  2. Anterior and posterior corneal stroma elasticity assessed using nanoindentation

    PubMed Central

    Dias, Janice; Ziebarth, Noël M.

    2013-01-01

    Corneal biomechanics is an essential parameter for developing diagnostic and treatment methods of corneal-related diseases. It is widely accepted that corneal mechanical strength stems from the stroma's collagenous composition. However, more comprehensive insight into the mechanical properties within the stroma is needed to improve current corneal diagnostic and treatment techniques. The purpose of this study was to perform elasticity characterization of anterior and posterior stromal regions of human corneas using atomic force microscopy (AFM). Nine pairs of human whole globes were placed in 20% Dextran solution, cornea side down, to restore the corneal thickness to physiological levels (400-600μm). The epithelium and Bowman's membrane were removed from all eyes. Anterior stromal AFM elasticity testing was then performed on left (OS) eyes. Additional stroma was removed from right (OD) eyes to allow posterior stromal measurements at a depth of 50% of the original thickness. All experiments were performed with corneas submerged in 15% Dextran to maintain corneal hydration. The results of the study showed that the Young's modulus of elasticity of the anterior stroma (average: 281 ± 214kPa; range: 59-764kPa) was significantly higher than that of the posterior stroma (average: 89.5 ± 46.1kPa; range: 29-179kPa) (p=0.014). In addition, a linear relationship was found between the posterior stromal elasticity and anterior stromal elasticity (p=0.0428). On average, the elasticity of the posterior stroma is 39.3% of the anterior stroma. In summary, there appears to be an elasticity gradient within the corneal stroma, which should be considered in the design and development of corneal diagnostic and treatment methods to enhance efficacy. PMID:23800511

  3. UVB light regulates expression of antioxidants and inflammatory mediators in human corneal epithelial cells

    PubMed Central

    Black, Adrienne T.; Gordon, Marion. K.; Heck, Diane E.; Gallo, Michael A.; Laskin, Debra L.; Laskin, Jeffrey D.

    2011-01-01

    The cornea is highly sensitive to ultraviolet B (UVB) light-induced oxidative stress, a process that results in the production of inflammatory mediators which have been implicated in tissue injury. In the present studies, we characterized the inflammatory response of human corneal epithelial cells to UVB (2.5 - 25 mJ/cm2). UVB caused a dose-dependent increase in the generation of reactive oxygen species in the cells. This was associated with increases in mRNA expression of the antioxidants Cu,Zn superoxide dismutase (SOD), Mn-SOD, catalase and heme oxygenase-1 (HO-1), as well as the glutathione S-transferases (GST), GSTA1-2, GSTA3, GSTA4, GSTM1, mGST2. UVB also upregulated expression of the proinflammatory cytokines, IFNγ, IL-1β, TGFβ and TNFα, and enzymes important in prostaglandin (PG) biosynthesis including cyclooxygenase-2 (COX-2) and the PG synthases mPGES-2, PGDS, PGFS and thromboxane synthase, and in leukotriene biosynthesis including 5-lipoxygenase (5-LOX), and the epidermal and platelet forms of 12-LOX and 15-LOX-2. UVB was found to activate JNK and p38 MAP kinases in corneal epithelial cells; ERK1/2 MAP kinase is constitutively active, but its activity was increased following UVB treatment. Inhibition of p38 blocked UVB-induced expression of TNFα, COX-2, PGDS and 15-LOX-2, while JNK inhibition suppressed TNFα and HO-1. These data indicate that UVB modulates corneal epithelial cell expression of antioxidants and proinflammatory mediators by distinct mechanisms. Alterations in expression of these mediators are likely to be important in regulating inflammation and protecting the cornea from UVB-induced oxidative stress. PMID:21300015

  4. Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser.

    PubMed

    Jumelle, Clotilde; Mauclair, Cyril; Houzet, Julien; Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Peoc'h, Michel; Acquart, Sophie; Gain, Philippe; Thuret, Gilles

    2015-01-01

    Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3-57.3) of CECs with low mortality (0.5%, range (0.55-1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for future

  5. Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser

    PubMed Central

    Jumelle, Clotilde; Mauclair, Cyril; Houzet, Julien; Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Peoc’h, Michel; Acquart, Sophie; Gain, Philippe; Thuret, Gilles

    2015-01-01

    Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3–57.3) of CECs with low mortality (0.5%, range (0.55–1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for

  6. Augmentation of arginase Ⅱ expression in the human endometrial epithelium in the secretory phase.

    PubMed

    Tajima, Makiko; Harada, Tatsuya; Ishikawa, Tomonori; Iwahara, Yuki; Kubota, Toshiro

    2012-12-03

    L-arginine is the common substrate for arginase and nitric oxide synthase (NOS). Arginase converts L-arginine to urea and L-ornithine. L-Ornithine is the principal precursor for the production of polyamines and L-proline, which are required for cell proliferation and collagen synthesis. Endothelial NOS is expressed in the human endometrial glandular epithelium, but the expression and physiological roles of arginase in the human endometrium are not clear. The objective of this study was to investigate the expression and distribution patterns of arginases Ⅰ (A-Ⅰ) and Ⅱ (A-Ⅱ) in the human endometrium by using immunohistochemistry, reverse transcription-polymerase chain reaction (RTPCR), and western blotting. A-Ⅰ and A-Ⅱ were detected by immunohistochemistry in human endometrial epithelial cells during the proliferative and secretory phases of the menstrual cycle. RT-PCR showed that A-Ⅰ and A-Ⅱ mRNA were expressed in human endometrial tissue. Western blotting analysis results showed the expression of A-Ⅱ protein. Immunohistochemistry and western blotting results showed that expression levels of A-Ⅱ were significantly higher in the secretory phase than in the proliferative phase. Increased A-Ⅱ levels in the secretory phase may be responsible for endometrial growth by increasing polyamines and proline products.

  7. DNA strand breaks in human nasal respiratory epithelium are induced upon exposure to urban pollution.

    PubMed Central

    Calderon-Garciduenas, L; Osnaya-Brizuela, N; Ramirez-Martinez, L; Villarreal-Calderon, A

    1996-01-01

    All organisms have the ability to respond and adapt to a myriad of environmental insults. The human respiratory epithelium, when exposed to oxidant gases in photochemical smog, is at risk of DNA damage and requires efficient cellular adaptative responses to resist the environmentally induced cell damage. Ozone and its reaction products induce in vitro and in vivo DNA single strand breaks (SSBs) in respiratory epithelial cells and alveolar macrophages. To determine if exposure to a polluted atmosphere with ozone as the main criteria pollutant induces SSBs in nasal epithelium, we studied 139 volunteers, including a control population of 19 children and 13 adult males who lived in a low-polluted Pacific port, 69 males and 16 children who were permanent residents of Southwest Metropolitan Mexico City (SWMMC), and 22 young males newly arrived to SWMMC and followed for 12 weeks. Respiratory symptoms, nasal cytology and histopathology, cell viabilities, and single-cell gel electrophoresis were investigated. Atmospheric pollutant data were obtained from a fixed-site monitoring station. SWMMC volunteers spent >7 hr/day outdoors and all had upper respiratory symptoms. A significant difference in the numbers of DNA-damaged nasal cells was observed between control and chronically exposed subjects, both in children (p<0.00001) and in adults (p<0.01). SSBs in newly arrived subjects quickly increased upon arrival to the city, from 39.8 +/- 8.34% in the first week to 67.29 +/- 2.35 by week 2. Thereafter, the number of cells with SSBs remained stable in spite of the continuous increase in cumulative ozone, suggesting a threshold for cumulative DNA nasal damage. Exposure to a polluted urban atmosphere induces SSBs in human nasal respiratory epithelium, and nasal SSBs could serve as a biomarker of ozone exposure. Further, because DNA strand breaks are a threat to cell viability and genome integrity and appear to be a critical lesion responsible for p53 induction, nasal SSBs should be

  8. DNA strand breaks in human nasal respiratory epithelium are induced upon exposure to urban pollution.

    PubMed

    Calderon-Garciduenas, L; Osnaya-Brizuela, N; Ramirez-Martinez, L; Villarreal-Calderon, A

    1996-02-01

    All organisms have the ability to respond and adapt to a myriad of environmental insults. The human respiratory epithelium, when exposed to oxidant gases in photochemical smog, is at risk of DNA damage and requires efficient cellular adaptative responses to resist the environmentally induced cell damage. Ozone and its reaction products induce in vitro and in vivo DNA single strand breaks (SSBs) in respiratory epithelial cells and alveolar macrophages. To determine if exposure to a polluted atmosphere with ozone as the main criteria pollutant induces SSBs in nasal epithelium, we studied 139 volunteers, including a control population of 19 children and 13 adult males who lived in a low-polluted Pacific port, 69 males and 16 children who were permanent residents of Southwest Metropolitan Mexico City (SWMMC), and 22 young males newly arrived to SWMMC and followed for 12 weeks. Respiratory symptoms, nasal cytology and histopathology, cell viabilities, and single-cell gel electrophoresis were investigated. Atmospheric pollutant data were obtained from a fixed-site monitoring station. SWMMC volunteers spent >7 hr/day outdoors and all had upper respiratory symptoms. A significant difference in the numbers of DNA-damaged nasal cells was observed between control and chronically exposed subjects, both in children (p<0.00001) and in adults (p<0.01). SSBs in newly arrived subjects quickly increased upon arrival to the city, from 39.8 +/- 8.34% in the first week to 67.29 +/- 2.35 by week 2. Thereafter, the number of cells with SSBs remained stable in spite of the continuous increase in cumulative ozone, suggesting a threshold for cumulative DNA nasal damage. Exposure to a polluted urban atmosphere induces SSBs in human nasal respiratory epithelium, and nasal SSBs could serve as a biomarker of ozone exposure. Further, because DNA strand breaks are a threat to cell viability and genome integrity and appear to be a critical lesion responsible for p53 induction, nasal SSBs should be

  9. [Micronucleus test of human oral buccal epithelium: problems, progress and prospects].

    PubMed

    Kalaev, V N; Artiukhov, V G; Nechaeva, M S

    2014-01-01

    The articles by russian and foreign authors for the period from 2000 to 2012, devoted to the problems of application, analysis and interpretation of the results of micronucleus test in human buccal epithelium has been analyzed in the review. Nuclear abnormality founding in the cells of the oral mucosa has been described. The paper summarizes works devoted to the analysis of the influence of the micronucleus test methods (painting, taking scrapings) to its results. Modern opinions about the factors of different etiology (sex, age, genotype, psycho-physiological characteristics, immune status, diseases of different etiology, man-made pollution, climatic and geographical conditions, ionizing and nonionizing radiation, chemical compounds (drugs, dietary supplements, androgenic steroids, etc.), dental fillings, occupational exposures, alcohol, using tobacco blends) inducing the estimation of nuclear aberration has been summarized as a scheme. The problems and unresolved issues related to the peculiarities of micronucleus test has been noted.

  10. Comparative gene expression study and pathway analysis of the human iris- and the retinal pigment epithelium

    PubMed Central

    ten Brink, Jacoline B.; Moerland, Perry D.; Heine, Vivi M.; Bergen, Arthur A.

    2017-01-01

    Background The retinal pigment epithelium (RPE) is a neural monolayer lining the back of the eye. Degeneration of the RPE leads to severe vision loss in, so far incurable, diseases such as age-related macular degeneration and some forms of retinitis pigmentosa. A promising future replacement therapy may be autologous iris epithelial cell transdifferentiation into RPE in vitro and, subsequently, transplantation. In this study we compared the gene expression profiles of the iris epithelium (IE) and the RPE. Methods We collected both primary RPE- and IE cells from 5 freshly frozen human donor eyes, using respectively laser dissection microscopy and excision. We performed whole-genome expression profiling using 44k Agilent human microarrays. We investigated the gene expression profiles on both gene and functional network level, using R and the knowledge database Ingenuity. Results The major molecular pathways related to the RPE and IE were quite similar and yielded basic neuro-epithelial cell functions. Nonetheless, we also found major specific differences: For example, genes and molecular pathways, related to the visual cycle and retinol biosynthesis are significantly higher expressed in the RPE than in the IE. Interestingly, Wnt and aryl hydrocarbon receptor (AhR-) signaling pathways are much higher expressed in the IE than in the RPE, suggesting, respectively, a possible pluripotent and high detoxification state of the IE. Conclusions This study provides a valuation of the similarities and differences between the expression profiles of the RPE and IE. Our data combined with that of the literature, represent a most comprehensive perspective on transcriptional variation, which may support future research in the development of therapeutic transplantation of IE. PMID:28827822

  11. Comparative gene expression study and pathway analysis of the human iris- and the retinal pigment epithelium.

    PubMed

    Bennis, Anna; Ten Brink, Jacoline B; Moerland, Perry D; Heine, Vivi M; Bergen, Arthur A

    2017-01-01

    The retinal pigment epithelium (RPE) is a neural monolayer lining the back of the eye. Degeneration of the RPE leads to severe vision loss in, so far incurable, diseases such as age-related macular degeneration and some forms of retinitis pigmentosa. A promising future replacement therapy may be autologous iris epithelial cell transdifferentiation into RPE in vitro and, subsequently, transplantation. In this study we compared the gene expression profiles of the iris epithelium (IE) and the RPE. We collected both primary RPE- and IE cells from 5 freshly frozen human donor eyes, using respectively laser dissection microscopy and excision. We performed whole-genome expression profiling using 44k Agilent human microarrays. We investigated the gene expression profiles on both gene and functional network level, using R and the knowledge database Ingenuity. The major molecular pathways related to the RPE and IE were quite similar and yielded basic neuro-epithelial cell functions. Nonetheless, we also found major specific differences: For example, genes and molecular pathways, related to the visual cycle and retinol biosynthesis are significantly higher expressed in the RPE than in the IE. Interestingly, Wnt and aryl hydrocarbon receptor (AhR-) signaling pathways are much higher expressed in the IE than in the RPE, suggesting, respectively, a possible pluripotent and high detoxification state of the IE. This study provides a valuation of the similarities and differences between the expression profiles of the RPE and IE. Our data combined with that of the literature, represent a most comprehensive perspective on transcriptional variation, which may support future research in the development of therapeutic transplantation of IE.

  12. DNA strand breaks in human nasal respiratory epithelium are induced upon exposure to urban pollution

    SciTech Connect

    Calderon-Garciduenas, L.; Osnaya-Brizuela, N.; Ramirez-Martinez, L.

    1996-02-01

    All organisms have the ability to respond and adapt to a myriad of environmental insults. The human respiratory epithelium, when exposed to oxidant gases in photochemical smog, is at risk of DNA damage and requires efficient cellular adaptative responses to resist the environmentally induced cell damage. Ozone and its reaction products induce in vitro and in vivo DNA single strand breaks (SSBs) in respiratory epithelial cells and alveolar macrophages. To determine if exposure to a polluted atmosphere with ozone as the main criteria pollutant of 19 children and 13 adult males who lived in a low-polluted Pacific port, 69 males and 16 children who were permanent residents of Southwest Metropolitan Mexico City (SWMMC), and 22 young males newly arrived to SWMMC and followed for 12 weeks. Respiratory symptoms, nasal cytology and histopathology, cell viabilities, and single-cell gel electrophoresis were investigated. Atmospheric pollutant data were obtained from a fixed-site monitoring station. SWMMC volunteers spent >7 hr/day outdoors and all had upper respiratory symptoms. A significant difference in the numbers of DNA-damaged nasal cells was observed between control and chronically exposed subjects, both in children (p<0.00001) and in adults (p>0.01). SSBs in newly arrived subjects quickly increased upon arrival to the city, from 39.8 {+-}8.34% in the first week to 67.29 {+-}2.35 by week 2. Thereafter, the number of cells with SSBs remained stable in spite of the continuous increase in cumulative ozone, suggesting a threshold for cumulative DNA nasal damage. Exposure to a polluted urban atmosphere induces SSBs in human nasal respiratory epithelium, and nasal SSBs could serve as a biomarker of ozone exposure. Further, because DNA strand breaks are a threat to cell viability and genome integrity and appear to be a critical lesion responsible for p53 induction, nasal SSBs should be evaluated in ozone-exposed individuals. 43 refs., 5 figs., 4 tabs.

  13. Excretion of Human Immunodeficiency Virus Type 1 through Polarized Epithelium by Immunoglobulin A▿

    PubMed Central

    Wright, Alison; Lamm, Michael E.; Huang, Yung T.

    2008-01-01

    Human immunodeficiency virus (HIV) is transmitted primarily sexually across mucosal surfaces. After infection, HIV propagates initially in the lamina propria below the polarized epithelium and causes extensive destruction of mucosal T cells. Immunoglobulin A (IgA) antibodies, produced in the lamina propria and then transcytosed across the mucosal epithelium into the lumen, can be the first line of immune defense against HIV. Here, we used IgA monoclonal antibodies against HIV envelope proteins to investigate the abilities of polarized primate and human epithelial cells to excrete HIV virions from the basolateral to the apical surface via polymeric Ig receptor (pIgR)-mediated binding and the internalization of HIV-IgA immune complexes. African green monkey kidney cells expressing pIgR demonstrated HIV excretion that was dependent on the IgA concentration and the exposure time. Matched IgG antibodies with the same variable regions as the IgA antibodies and IgA antibodies to non-HIV antigens had no HIV excretory function. A mixture of two IgA anti-bodies against gp120 and gp41 showed a synergistic increase in the level of HIV excreted. The capacity for HIV excretion correlated with the ability of IgA antibodies to bind HIV and of the resulting immune complexes to bind pIgR. Consistent with the epithelial transcytosis of HIV-IgA immune complexes, the colocalization of HIV proteins and HIV-specific IgA was detected intracellularly by confocal microscopy. Our results suggest the potential of IgA antibodies to excrete HIV from mucosal lamina propria, thereby decreasing the viral burden, access to susceptible cells, and the chronic activation of the immune system. PMID:18829757

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

    PubMed

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

    2015-11-08

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

  15. Amniotic membrane transplantation for the management of corneal epithelial defects: an in vivo confocal microscopic study.

    PubMed

    Nubile, M; Dua, H S; Lanzini, T Evb-M; Carpineto, P; Ciancaglini, M; Toto, L; Mastropasqua, L

    2008-01-01

    To evaluate amniotic membrane (AM) tissue morphology and corneal epithelial healing in human eyes after amniotic membrane transplantation (AMT), using laser scanning in vivo confocal microscopy (IVCM). Twenty eyes of 20 patients, treated with single layer epithelial side up AMT for chemical burns in the acute stage (n = 8) and persistent corneal epithelial defect (n = 12) were studied by serial IVCM post-AMT until complete re-epithelisation. Changes in morphology of transplanted amniotic tissue and healing corneal epithelium were noted. AM and corneal epithelial cell density was calculated using image-analysis software. IVCM enabled visualisation of transplanted AM and of regenerating epithelial cells under the AM. The mean AM epithelial cell density, 1 day after transplant, was 4613 (SD 380) cells/mm2. The average AM epithelial thickness was 35 (4) microm, while the AM stromal thickness was 116 (31) microm. The amniotic stroma appeared to be composed of a superficial dense fibrous layer and a deeper loose reticular network of fibres. Amniotic epithelium was lost within 15 days of transplant, and complete re-epithelisation of the corneal surface was achieved between 1 and 4 weeks. Laser scanning IVCM is a useful method for evaluating AM tissue morphology, degradation and corneal epithelial healing after AMT for different clinical indications. When the amniotic membrane acts as a patch, that is epithelial cells migrate under rather than over the membrane, the membrane disintegrates and is lost.

  16. Inhibition of mouse alkali burn induced-corneal neovascularization by recombinant adenovirus encoding human vasohibin-1

    PubMed Central

    Xie, Zhao-lian; Xiao, Ou; Yang, Xiao-ru; Heng, Boon Chin; Sato, Yasufumi

    2010-01-01

    Purpose To evaluate the activity of recombinant adenovirus encoding human vasohibin-1 (Ad-Vasohibin-1) on mouse corneal neovasularization induced by alkali burn. Methods For the treatment group, 50 mice each received subconjunctival injection (5 μl) of 109 plaque forming units of replication-defective Ad-Vasohibin-1. Control group mice received the same dosage of blank adenoviral vector (AdNull). Five days after injection, corneal neovascularization (CNV) was induced by placing 2.5 μl of 0.1 M NaOH on the right cornea for 30 s. Subsequently, CNV was observed and photographed every 3 days for a total duration of 9 days after the alkali burn. The percentage of neovascularized area was measured and compared with the AdNull control. The expression of human vasohibin-1 protein was detected by immunohistochemistry and western blotting at 5, 8, and 14 days after injection. The mRNA expression levels of murine vascular endothelial growth factor (Vegf), VEGF receptor 1 and 2 (Vegfr1, Vegfr2), and vasohibin-1 (Vash1) were analyzed and compared by real time quantitative reverse-transcription polymerase chain reaction. Results The percentage of neovascularized area within the cornea was significantly reduced in mice treated with Ad-Vasohibin-1 compared to mice treated with AdNull at every time point after alkali-induced injury (7.11%±3.91% and 15.48%±1.79% of corneal area in the treatment and control groups, respectively, on day 3; 31.64%±4.71% and 43.93%±6.15% on day 6, and 45.02%±9.98% and 66.24%±7.17% on day 9, all p<0.001). Human vasohibin-1 protein was detected at the injection sites on day 3 after corneal burn and was highly expressed in the central subepithelial stroma and co-localized with neovascularized vessels within the alkali-treated cornea on day 6. On day 9, the peripheral cornea exhibited a similar staining pattern as the central cornea, but a more intense vasohibin-1 immunostaining signal was detected in the deep stroma. Some of the vasohibin-1 stain

  17. Three-dimensional arrangement of elastic fibers in the human corneal stroma.

    PubMed

    Lewis, Philip N; White, Tomas L; Young, Robert D; Bell, James S; Winlove, C Peter; Meek, Keith M

    2016-05-01

    The cornea is the main refracting lens in the eye. As part of the outer tunic it has to be resilient, a property conferred by the organisation of the constituent collagen. It also has to be sufficiently elastic to regain its exact shape when deformed, in order not to distort the retinal image. The basis of this elasticity is not fully understood. The purpose of this study was to characterise in three dimensions the arrangement and distribution of elastic fibers in the human corneal stroma, using serial block face scanning electron microscopy. We have demonstrated that there exists a complex network of elastic fibers that appear to originate in the sclera or limbus. These appear as elastic sheets in the limbus and peripheral cornea immediately above the trabecular meshwork which itself appears to extend above Descemet's membrane in the peripheral stroma. From these sheets, elastic fibers extend into the cornea; moving centrally they bifurcate and trifurcate into narrower fibers and are concentrated in the posterior stroma immediately above Descemet's membrane. We contend that elastic sheets will play an important role in the biomechanical deformation and recovery of the peripheral cornea. The network may also have practical implications for understanding the structural basis behind a number of corneal surgeries.

  18. Human corneal limbal epithelial cell response to varying silk film geometric topography in vitro.

    PubMed

    Lawrence, Brian D; Pan, Zhi; Liu, Aihong; Kaplan, David L; Rosenblatt, Mark I

    2012-10-01

    Silk fibroin films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding silk films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that silk film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment and focal adhesion (FA) localization. Most notably, parallel line patterned surfaces displayed a 36-54% increase on average in initial cell attachment, which corresponded to a more than 2-fold increase in FA localization when compared to other silk film surfaces and controls. In addition, distinct localization of FA formation was observed along the edges for all patterned silk film topographies. In conclusion, silk film feature topography appears to help direct corneal epithelial cell response and cytoskeleton development, especially with regard to FA distribution, in vitro.

  19. Human β-NGF gene transferred to cat corneal endothelial cells.

    PubMed

    Luo, Wen-Juan; Liu, Min; Zhao, Gui-Qiu; Wang, Chuan-Fu; Hu, Li-Ting; Liu, Xiang-Ping

    2016-01-01

    To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco's modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result showed that the

  20. Human β-NGF gene transferred to cat corneal endothelial cells

    PubMed Central

    Luo, Wen-Juan; Liu, Min; Zhao, Gui-Qiu; Wang, Chuan-Fu; Hu, Li-Ting; Liu, Xiang-Ping

    2016-01-01

    AIM To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. METHODS The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco's modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. RESULTS The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result

  1. The modelling of the influence of a corneal geometry on the pupil image of the human eye

    NASA Astrophysics Data System (ADS)

    Szczesna, D. H.; Kasprzak, H. T.

    2006-07-01

    In normal conditions, a pupil of the eye is observed through the optical system of the cornea. The cornea is the anterior surface of the eye and is the major refractive element of the human eye. The influence of the corneal shape should not be neglected in measurements of the pupil size. The purpose of this study was to estimate the influence of the corneal geometry, the diameter of the pupil and its position in the anterior chamber on the magnification and position of the image of the pupil. The numerical calculations presented in the paper assume infinitely thin cornea, and the corneal topography was approximated by the elongated ellipsoid. The ray tracing procedure was used in our numerical modelling. The magnification of the pupil image amounted to about 10% and increases with decrease of radius of curvature and eccentricity of the corneal profile and decreases for the largest pupil. The results show also that the pupil image is placed nearer the corneal apex than the real pupil. The image of the pupil is always blurred, which limits the sharp observation of the pupil.

  2. Somatostatin receptor activation (sst(1) -sst(5) ) differentially influences human retinal pigment epithelium cell viability.

    PubMed

    Papadaki, Thekla; Tsilimbaris, Miltiadis; Pallikaris, Ioannis; Thermos, Kyriaki

    2010-09-01

    To investigate the differential effects of somatostatin and its receptors (sst(1-5) ) on the viability of cultured human retinal pigment epithelium (hRPE) cells. MTT [3 (4, 5-dimethylthiazol-2yl)-2, 5 diphenyltetrazolium bromide], APO Percentage(TM) and trypan blue assays were performed to assess the mechanisms via which somatostatin (10(-10) -10(-4) m) and selective receptor (sst(1-5) ) ligands (10(-12) -10(-4) m) affect cell viability. The effect of orthovanadate (phosphatase inhibitor, 10(-7) -10(-5) m) on somatostatin's (10(-5) m) actions was examined, and western blot analysis was employed to determine the presence of ssts and phosphotyrosine phosphatase SHP-1 in human RPE cells. Somatostatin and selective ligands for the five somatostatin receptor subtypes (sst(1-5) ) decreased cell viability in a concentration-dependent manner. The observed decrease in cell number was partly because of apoptosis via the activation of sst(1) and sst(5) receptors. Activation of sst(2) , sst(3) and sst(4) receptors led to inhibition of cell growth that did not involve apoptosis, but rather antiproliferative actions. SHP-1 was found in the human RPE cells and sodium orthovanadate reversed somatostatin's actions. This study provides new information regarding the involvement of ssts in human RPE cell viability and suggests that a pathway involving the phosphotyrosine phosphatase may mediate somatostatin's actions. © 2010 The Authors. Journal compilation © 2010 Acta Ophthalmol.

  3. Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells.

    PubMed

    Hirayama, Masatoshi; Ko, Shigeru B H; Kawakita, Tetsuya; Akiyama, Tomohiko; Goparaju, Sravan K; Soma, Atsumi; Nakatake, Yuhki; Sakota, Miki; Chikazawa-Nohtomi, Nana; Shimmura, Shigeto; Tsubota, Kazuo; Ko, Minoru S H

    2017-01-01

    Dry eye disease is the most prevalent pathological condition in aging eyes. One potential therapeutic strategy is the transplantation of lacrimal glands, generated in vitro from pluripotent stem cells such as human embryonic stem cells, into patients. One of the preceding requirements is a method to differentiate human embryonic stem cells into lacrimal gland epithelium cells. As the first step for this approach, this study aims to identify a set of transcription factors whose overexpression can promote the differentiation of human embryonic stem cells into lacrimal gland epithelium-like cells. We performed microarray analyses of lacrimal glands and lacrimal glands-related organs obtained from mouse embryos and adults, and identified transcription factors enriched in lacrimal gland epithelium cells. We then transfected synthetic messenger RNAs encoding human orthologues of these transcription factors into human embryonic stem cells and examined whether the human embryonic stem cells differentiate into lacrimal gland epithelium-like cells by assessing cell morphology and marker gene expression. The microarray analysis of lacrimal glands tissues identified 16 transcription factors that were enriched in lacrimal gland epithelium cells. We focused on three of the transcription factors, because they are expressed in other glands such as salivary glands and are also known to be involved in the development of lacrimal glands. We tested the overexpression of various combinations of the three transcription factors and PAX6, which is an indispensable gene for lacrimal glands development, in human embryonic stem cells. Combining PAX6, SIX1, and FOXC1 caused significant changes in morphology, i.e., elongated cell shape and increased expression (both RNAs and proteins) of epithelial markers such as cytokeratin15, branching morphogenesis markers such as BARX2, and lacrimal glands markers such as aquaporin5 and lactoferrin. We identified a set of transcription factors enriched in

  4. The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

    PubMed

    Pathak, Meeta; Olstad, O K; Drolsum, Liv; Moe, Morten C; Smorodinova, Natalia; Kalasova, Sarka; Jirsova, Katerina; Nicolaissen, Bjørn; Noer, Agate

    2016-12-01

    Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene

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

    PubMed

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

    2016-10-14

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

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

    PubMed

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

    2017-03-01

    Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Stem Cells Translational Medicine 2017;6:761-766. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  7. Geometrical Custom Modeling of Human Cornea In Vivo and Its Use for the Diagnosis of Corneal Ectasia

    PubMed Central

    Cavas-Martínez, Francisco; Fernández-Pacheco, Daniel G.; De la Cruz-Sánchez, Ernesto; Nieto Martínez, José; Fernández Cañavate, Francisco J.; Vega-Estrada, Alfredo; Plaza-Puche, Ana B.; Alió, Jorge L.

    2014-01-01

    Aim To establish a new procedure for 3D geometric reconstruction of the human cornea to obtain a solid model that represents a personalized and in vivo morphology of both the anterior and posterior corneal surfaces. This model is later analyzed to obtain geometric variables enabling the characterization of the corneal geometry and establishing a new clinical diagnostic criterion in order to distinguish between healthy corneas and corneas with keratoconus. Method The method for the geometric reconstruction of the cornea consists of the following steps: capture and preprocessing of the spatial point clouds provided by the Sirius topographer that represent both anterior and posterior corneal surfaces, reconstruction of the corneal geometric surfaces and generation of the solid model. Later, geometric variables are extracted from the model obtained and statistically analyzed to detect deformations of the cornea. Results The variables that achieved the best results in the diagnosis of keratoconus were anterior corneal surface area (ROC area: 0.847, p<0.000, std. error: 0.038, 95% CI: 0.777 to 0.925), posterior corneal surface area (ROC area: 0.807, p<0.000, std. error: 0.042, 95% CI: 0,726 to 0,889), anterior apex deviation (ROC area: 0.735, p<0.000, std. error: 0.053, 95% CI: 0.630 to 0.840) and posterior apex deviation (ROC area: 0.891, p<0.000, std. error: 0.039, 95% CI: 0.8146 to 0.9672). Conclusion Geometric modeling enables accurate characterization of the human cornea. Also, from a clinical point of view, the procedure described has established a new approach for the study of eye-related diseases. PMID:25329896

  8. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.

    PubMed Central

    Mason, S. J.; Paradiso, A. M.; Boucher, R. C.

    1991-01-01

    1 The role of extracellular nucleotides in regulation of ion transport activities (short circuit current, Isc) of human respiratory epithelia was studied. 2 Application of nucleotides to the apical or basolateral membrane of human nasal epithelium induced a concentration-dependent increase in Isc. 3 The rank order of potency of purine- or pyrimidine-induced changes in Isc of normal human nasal epithelium when applied to the apical membrane (UTP greater than or equal to ATP greater than ATP gamma S greater than 2MeSATP greater than ADP beta S much greater than beta gamma MeATP greater than or equal to alpha beta MeATP) or basolateral membrane (2MeSATP greater than UTP greater than ATP greater than ATP gamma S greater than alpha beta MeATP greater than beta gamma MeATP) is consistent with involvement of a P2 purinoceptor. A similar rank order of potencies was observed for nucleotide effects on intracellular calcium measured by Fura-2 fluorescence using microspectrofluorimetry. 4 Similar nucleotide potency in the regulation of ion transport and intracellular calcium in cystic fibrosis (CF) airway epithelium (UTP greater than or equal to ATP) was observed, suggesting purinoceptors might be used to stimulate ion transport processes that would promote hydration of airway secretions and facilitate their clearance from CF lungs. 5 These data provide evidence for the regulation of ion transport by P2 purinoceptors in normal and cystic fibrosis human airway epithelium. PMID:1718521

  9. Molecular Impact of Electronic Cigarette Aerosol Exposure in Human Bronchial Epithelium.

    PubMed

    Moses, Elizabeth; Wang, Teresa; Corbett, Sean; Jackson, George R; Drizik, Eduard; Perdomo, Catalina; Perdomo, Claudia; Kleerup, Eric; Brooks, Daniel; O'Connor, George; Dubinett, Steven; Hayden, Patrick; Lenburg, Marc E; Spira, Avrum

    2017-01-01

    Little evidence is available regarding the physiological effects of exposure to electronic cigarette (ECIG) aerosol. We sought to determine the molecular impact of ECIG aerosol exposure in human bronchial epithelial cells (HBECs). Gene-expression profiling was conducted in primary grown at air liquid interface and exposed to 1 of 4 different ECIG aerosols, traditional tobacco cigarette (TCIG) smoke, or clean air. Findings were validated experimentally with quantitative polymerase chain reaction and a reactive oxygen species immunoassay. Using gene set enrichment analysis, signatures of in vitro ECIG exposure were compared with those generated from bronchial epithelial brushings of current TCIG smokers and former TCIG smokers currently using ECIGs. We found 546 genes differentially expressed across the ECIG, TCIG, and air-exposed groups of HBECs (ANOVA; FDR q < .05; fold change > 1.5). A subset of these changes were shared between TCIG- and ECIG-exposed HBECs. ECIG exposure induced genes involved in oxidative and xenobiotic stress pathways and increased a marker of reactive oxygen species production in a dose-dependent manner. ECIG exposure decreased expression of genes involved in cilia assembly and movement. Furthermore, gene-expression differences observed in vitro were concordant with differences observed in airway epithelium collected from ECIG users (q < .01). In summary, our data suggest that ECIG aerosol can induce gene-expression changes in bronchial airway epithelium in vitro, some of which are shared with TCIG smoke. These changes were generally less pronounced than the effects of TCIG exposure and were more pronounced in ECIG products containing nicotine than those without nicotine. Our data further suggest that the gene-expression alterations seen with the in vitro exposure system reflects the physiological effects experienced in vivo by ECIG users.

  10. Basal cells as stem cells of the mouse trachea and human airway epithelium

    PubMed Central

    Rock, Jason R.; Onaitis, Mark W.; Rawlins, Emma L.; Lu, Yun; Clark, Cheryl P.; Xue, Yan; Randell, Scott H.; Hogan, Brigid L. M.

    2009-01-01

    The pseudostratified epithelium of the mouse trachea and human airways contains a population of basal cells expressing Trp-63 (p63) and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreERT2 transgenic mouse line for lineage tracing, we show that basal cells generate differentiated cells during postnatal growth and in the adult during both steady state and epithelial repair. We have fractionated mouse basal cells by FACS and identified 627 genes preferentially expressed in a basal subpopulation vs. non-BCs. Analysis reveals potential mechanisms regulating basal cells and allows comparison with other epithelial stem cells. To study basal cell behaviors, we describe a simple in vitro clonal sphere-forming assay in which mouse basal cells self-renew and generate luminal cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, ITGA6 and NGFR, which can be used in combination to purify human lung basal cells by FACS. Like those from the mouse trachea, human airway basal cells both self-renew and generate luminal daughters in the sphere-forming assay. PMID:19625615

  11. Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors

    PubMed Central

    Teixeira, Vitor H; Nadarajan, Parthiban; Graham, Trevor A; Pipinikas, Christodoulos P; Brown, James M; Falzon, Mary; Nye, Emma; Poulsom, Richard; Lawrence, David; Wright, Nicholas A; McDonald, Stuart; Giangreco, Adam; Simons, Benjamin D; Janes, Sam M

    2013-01-01

    Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to “neutral drift” of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease. DOI: http://dx.doi.org/10.7554/eLife.00966.001 PMID:24151545

  12. Elastic modulus and collagen organization of the rabbit cornea: epithelium to endothelium

    PubMed Central

    Thomasy, Sara M.; Krishna Raghunathan, Vijay; Winkler, Moritz; Reilly, Christopher M.; Sadeli, Adeline R.; Russell, Paul; Jester, James V.; Murphy, Christopher J.

    2013-01-01

    The rabbit is commonly used to evaluate new corneal prosthetics and study corneal wound healing. Knowledge of the stiffness of the rabbit cornea would better inform design and fabrication of keratoprosthetics and substrates with relevant mechanical properties for in vitro investigations of corneal cellular behavior. This study determined the elastic modulus of the rabbit corneal epithelium, anterior basement membrane (ABM), anterior and posterior stroma, Descemet’s membrane (DM) and endothelium using atomic force microscopy (AFM). In addition, three-dimensional collagen fiber organization of the rabbit cornea was determined using nonlinear optical high-resolution macroscopy. Elastic modulus as determined by AFM for each corneal layer was: epithelium 0.57 ± 0.29 kPa (mean ± SD), ABM 4.5 ± 1.2 kPa, anterior stroma 1.1 ± 0.6 kPa, posterior stroma 0.38 ± 0.22 kPa, DM 11.7 ± 7.4 kPa, and endothelium 4.1 ± 1.7 kPa. Biophysical properties, including elastic modulus, are unique for each layer of the rabbit cornea and are dramatically softer in comparison to the corresponding regions of the human cornea. Collagen fiber organization is also dramatically different between the two species with markedly less intertwining observed in the rabbit versus human cornea. Given that substratum stiffness considerably alters corneal cell behavior, keratoprosthetics that incorporate mechanical properties simulating the native human cornea may not elicit optimal cellular performance in rabbit corneas that have dramatically different elastic moduli. These data will allow for the design of substrates that better mimic the biomechanical properties of the corneal cellular environment. PMID:24084333

  13. Cell-Deposited Matrix Improves Retinal Pigment Epithelium Survival on Aged Submacular Human Bruch's Membrane

    PubMed Central

    Sugino, Ilene K.; Gullapalli, Vamsi K.; Sun, Qian; Wang, Jianqiu; Nunes, Celia F.; Cheewatrakoolpong, Noounanong; Johnson, Adam C.; Degner, Benjamin C.; Hua, Jianyuan; Liu, Tong; Chen, Wei; Li, Hong

    2011-01-01

    Purpose. To determine whether resurfacing submacular human Bruch's membrane with a cell-deposited extracellular matrix (ECM) improves retinal pigment epithelial (RPE) survival. Methods. Bovine corneal endothelial (BCE) cells were seeded onto the inner collagenous layer of submacular Bruch's membrane explants of human donor eyes to allow ECM deposition. Control explants from fellow eyes were cultured in medium only. The deposited ECM was exposed by removing BCE. Fetal RPE cells were then cultured on these explants for 1, 14, or 21 days. The explants were analyzed quantitatively by light microscopy and scanning electron microscopy. Surviving RPE cells from explants cultured for 21 days were harvested to compare bestrophin and RPE65 mRNA expression. Mass spectroscopy was performed on BCE-ECM to examine the protein composition. Results. The BCE-treated explants showed significantly higher RPE nuclear density than did the control explants at all time points. RPE expressed more differentiated features on BCE-treated explants than on untreated explants, but expressed very little mRNA for bestrophin or RPE65. The untreated young (<50 years) and African American submacular Bruch's membrane explants supported significantly higher RPE nuclear densities (NDs) than did the Caucasian explants. These differences were reduced or nonexistent in the BCE-ECM-treated explants. Proteins identified in the BCE-ECM included ECM proteins, ECM-associated proteins, cell membrane proteins, and intracellular proteins. Conclusions. Increased RPE survival can be achieved on aged submacular human Bruch's membrane by resurfacing the latter with a cell-deposited ECM. Caucasian eyes seem to benefit the most, as cell survival is the worst on submacular Bruch's membrane in these eyes. PMID:21398292

  14. Interocular comparison by in vivo confocal microscopy of the 2-dimensional architecture of the normal human corneal subbasal nerve plexus.

    PubMed

    Misra, Stuti; Craig, Jennifer P; McGhee, Charles N J; Patel, Dipika V

    2012-12-01

    To investigate the configuration of the living human corneal subbasal nerve plexus in paired eyes of normal subjects using in vivo confocal microscopy. Laser scanning in vivo confocal microscopy was performed on both corneas of healthy human subjects, and a grid fixation pattern facilitated examination of consistent areas of central to midperipheral cornea. Macromedia/Adobe Freehand 10 was used to manually arrange images into contiguous montages. The subbasal nerve density and overall patterns were analyzed. Both eyes of 6 subjects (3 women and 3 men, aged between 25 and 36 years) were examined. In all subjects, the subbasal nerve plexus exhibited a clockwise whorl configuration inferior to the central cornea. The mean subbasal nerve density at the whorl was 39.17±4.95 mm/mm and 41.36±4.19 mm/mm in the right and left eyes, respectively. There was no significant difference in the nerve density between the eyes (P=0.61). Bland and Altman analysis confirmed high intraobserver repeatability and moderate interobserver repeatability. This study reveals the marked similarities between the corneal subbasal nerve plexus configuration in the right and left eyes of the living human cornea, highlighting that the typical mirror-image symmetry in corneal topographic patterns is not obeyed in respect to corneal innervation and that a clockwise orientation of the subbasal plexus is typically encountered. There was no statistical difference in the subbasal nerve density between the eyes.

  15. Corneal modeling using conic section fits of PAR corneal topography system measurements

    NASA Astrophysics Data System (ADS)

    Zipper, Stanley; Manns, Fabrice; Fernandez, Viviana; Sandadi, Samith; Ho, Arthur; Parel, Jean-Marie A.

    2001-06-01

    The purpose of this study was to measure the average shape and variability of human corneas and to develop a tool for analyzing, height, curvature, and aberrations based on a conic section model. Fresh Eye Bank Eyes were placed in Dextran until the corneal thickness reached a physiological value. The eyes were placed in a custom made holder and measured using an intraoperative PAR Corneal Topography System (CTS) mounted on an operation microscope. Topography was measured before and after removal of the epithelium. A series of MATLAB functions were written to analyze the raw-z (height) data in polar coordinates. The functions fit conic sections to the PAR CTS data along hemi-meridians at 5 degree(s) intervals. The conic shape factor and apical radius were used to calculate and display the curvature. The dependence of these parameters with meridional position was examined.

  16. Particulate matter contamination in the corneal stroma of severe eye burns in humans.

    PubMed

    Schrage, N F; Reim, M; Burchard, W G

    1990-01-01

    Corneal buttons obtained from keratoplasty were examined by energy dispersive x-ray analysis (EDXA) combined with scanning electron microscopy (SEM). This method enables to assay the mineral composition of minute parts of tissue samples identified in SEM images. Samples were cut from paraffin embedded corneae, deparaffinized in xylol, dried in aceton, critical-point desiccated, covered by evaporating with a thin layer of carbon and examined by SEM. In healthy human donor eyes, only some iron particles had been found. In the 22 patients samples high amounts of different particles were identified, materials from rubber stoppers, chromesteel, titanium pigments, talcum, barium and glass. Furthermore a lot of different metal particles containing varying amounts of Na, Mg, Al, Si, P, S, Cl, K, Ca, Fe, Cu, Cr, Zn, La and Ce were detected. Some particles may be caused by the initial trauma, others by therapy. Such contaminations might have supported leucocyte and fibrocyte invasion increasing the inflammatory reaction in the burnt cornea.

  17. Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium.

    PubMed

    Beaulieu, Alexandre; Gravel, Émilie; Cloutier, Alexandre; Marois, Isabelle; Colombo, Éloïc; Désilets, Antoine; Verreault, Catherine; Leduc, Richard; Marsault, Éric; Richter, Martin V

    2013-04-01

    Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

  18. Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium

    PubMed Central

    Beaulieu, Alexandre; Gravel, Émilie; Cloutier, Alexandre; Marois, Isabelle; Colombo, Éloïc; Désilets, Antoine; Verreault, Catherine; Leduc, Richard; Marsault, Éric

    2013-01-01

    Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place. PMID:23365447

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

    PubMed Central

    Wen, Qian; Tian, Cheng-Lei

    2016-01-01

    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. PMID:27022135

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

    PubMed

    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.

  1. [Effect of various anti-glaucoma eyedrops on human corneal epithelial cells].

    PubMed

    Aoyama, Yumiko; Motoki, Masamitsu; Hashimoto, Mariko

    2004-02-01

    To investigate the effects of antiglaucoma eyedrops and vehicles on the proliferation of human corneal epithelial cells. Seven eyedrops[prostaglandin F2 alpha analogs(2), beta blockers(40), topical carbonic anhydrase inhibitor(1)], and six of the eyedrop vehicles, excluding that of Xalatan, were used. Anti-glaucoma eyedrops and vehicles were serially diluted 2-fold with culture medium(10-2,560 fold). The mixture was added to human corneal epithelial cells and incubated for 48 hrs. Cell proliferation was measured by commercial assay kit. Dye-reagents were added to the wells and incubated for 1 h at 37 degrees C. Optical density were measured at 490 nm. The dilution rate for 50% inhibition was calculated as the dilution rate of drugs or vehicles necessary to produce 50% inhibition of cell proliferation. All drugs completely inhibited cell proliferation when the dilution rate was low. At 40-fold dilution, Trusopt and Timoptol showed a significant decrease in cell growth inhibition. On the other hand, Rescula showed almost 100% inhibition at 160-fold dilution. Above 640-fold dilution, the inhibition rate of all drugs became 50% or less and there was no significant difference between drugs. Vehicles also inhibited cell growth. The dilution rates for growth inhibition by vehicles were different from those of drugs. The dilution rate at 50% inhibition of anti-glaucoma eyedrops decreased in the following order: Rescula > Xalatan > Betoptic > Hypadil > Mikelan > Timoptol > Trusopt. The dilution rate for 50% inhibition of vehicles decreased in the following order: Rescula vehicle > Hypadil vehicle > Betoptic vehicle > Mikelan vehicle > Timoptol vehicle > Trusopt vehicle. All anti-glaucoma eyedrops inhibited cell proliferation. These effects were stronger in prostaglandin F2 alpha analogs and weakest in Trusopt. Furthermore, the inhibition of cell proliferation was caused also by the vehicle of eyedrops, and the influence of the vehicle varied in each type of eyedrops.

  2. Generation of Human Corneal Endothelial Cells via In Vitro Ocular Lineage Restriction of Pluripotent Stem Cells

    PubMed Central

    Zhao, Jiagang J.; Afshari, Natalie A.

    2016-01-01

    Purpose We generate a renewable supply of corneal endothelial cells (CEC) from human pluripotent stem cells (PSCs) under defined culture conditions. Methods Corneal endothelial cell induction was driven by small molecules in a stepwise fashion of lineage specification. During the initial phase, PSC fate was restricted to the eye field-like state and became eye field stem cells (EFSCs). In the second phase, PSC-derived EFSCs were further directed toward either neural crest lineage or retinal lineage. The CECs were directly induced from ocular neural crest stem cells (NCSCs) by suppressing TGF-β and ROCK signaling. Results Under chemically defined conditions, PSCs were massively converted into EFSCs and subsequently NCSCs. Eye field cell identity was characterized by the expression of key fate restriction factors for early eye field cells, such as PAX6, LHX2, and VSX2. The induction of ocular NCSCs was initiated by promoting WNT signaling in EFSCs. Within 2 weeks of induction, the majority of cells expressed the typical neural crest markers p75NTR and HNK-1. Eye field stem cell-derived NCSCs can be propagated and cryopreserved. Subsequently, a CEC monolayer was induced from adherent NCSCs in the presence of small molecular inhibitors to suppress TGF-β and ROCK signaling. The polygon-shaped CEC-like cells became visible after a week in culture. The NCSC-derived CECs expressed typical CEC markers, such as N-Cadherin and Na+/K+-ATPase. Conclusions A novel small molecule-based approach was developed to derive human CECs from PSCs via ocular lineage specification. Moreover, EFSC-derived NCSCs could serve as an immediate source cell for rapid CEC induction in vitro. PMID:28002562

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

    PubMed

    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.

  4. Inhibition by rebamipide of cytokine-induced or lipopolysaccharide-induced chemokine synthesis in human corneal fibroblasts.

    PubMed

    Fukuda, Ken; Ishida, Waka; Tanaka, Hiroshi; Harada, Yosuke; Fukushima, Atsuki

    2014-12-01

    The dry-eye drug rebamipide has mucin secretagogue activity in and anti-inflammatory effects on corneal epithelial cells. Corneal stromal fibroblasts (transdifferentiated keratocytes) function as immune modulators in the pathogenesis of chronic ocular allergic inflammation and in innate immune responses at the ocular surface. The possible anti-inflammatory effects of rebamipide on human corneal stromal fibroblasts were examined. Serum-deprived cells were incubated for 1 h with rebamipide and then for various times in the additional absence or presence of cytokines or bacterial lipopolysaccharide (LPS). The release of chemokines into culture supernatants was determined with ELISAs. The intracellular abundance of chemokine mRNAs was quantitated by reverse transcription and real-time PCR analysis. Degradation of the nuclear factor κB (NFκB) inhibitor IκBα was detected by immunoblot analysis. Rebamipide suppressed the release of interleukin (IL)-8 and the upregulation of IL-8 mRNA induced by tumour necrosis factor α (TNF-α) or LPS in corneal fibroblasts. It also inhibited eotaxin-1 (CCL-11) expression at the protein and mRNA levels induced by the combination of TNF-α and IL-4. In addition, rebamipide attenuated the degradation of IκBα induced by TNF-α or LPS. Rebamipide inhibited the synthesis of chemokines by corneal fibroblasts in association with suppression of NFκB signalling. Rebamipide may therefore prove effective for the treatment of corneal stromal inflammation associated with allergy or bacterial infection. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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

    PubMed

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

    2013-04-17

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

  6. Differential expression of TYRP1 in adult human retinal pigment epithelium and uveal melanoma cells

    PubMed Central

    QIU, CHUN; LI, PENG; BI, JIANJUN; WU, QING; LU, LINNA; QIAN, GUANXIANG; JIA, RENBING; JIA, RONG

    2016-01-01

    Uveal melanoma (UM) is the most frequently occurring primary intraocular malignancy in adults. Tyrosinase (TYR) is a copper-containing enzyme and a type I membrane protein that is involved in the generation of melanin, the main pigment in vertebrates. TYR-related protein 1 (TYRP1) is regarded to have a crucial role in the immunotherapy of melanoma. As biomarkers, the TYR-related proteins, TYRP1 and TYRP2, exhibit specific expression in melanocytes, while also contributing to melanin synthesis within melanosomes. In the present study, the differential expression of TYRP1 was investigated at the mRNA, protein and morphological levels in four human UM cell lines (SP6.5, OM431, OCM1 and OCM290) and the human retinal pigment epithelium (RPE) cell line, using polymerase chain reaction, western blotting, immunocytochemistry and immunofluorescence staining. It was found that SP6.5 cells expressed the highest level of TYRP1, in comparison to SP6.5 OCM1 and OM431 cells, which produced less TYRP1, and OCM290 cells, which produced almost no TYRP1. No TYRP1 protein expression was identified in the RPE cell line. These findings indicate the potential use of TYRP1 in the development of therapy for UM. PMID:27073483

  7. A novel Bruch's membrane-mimetic electrospun substrate scaffold for human retinal pigment epithelium cells.

    PubMed

    Xiang, Ping; Wu, Kun-Chao; Zhu, Ying; Xiang, Lue; Li, Chong; Chen, Deng-Long; Chen, Feng; Xu, Guotong; Wang, Aijun; Li, Min; Jin, Zi-Bing

    2014-12-01

    Various artificial membranes have been used as scaffolds for retinal pigment epithelium cells (RPE) for monolayer reconstruction, however, long-term cell viability and functionality are still largely unknown. This study aimed to construct an ultrathin porous nanofibrous film to mimic Bruch's membrane, and in particular to investigate human RPE cell responses to the resultant substrates. An ultrathin porous nanofibrous membrane was fabricated by using regenerated wild Antheraea pernyi silk fibroin (RWSF), polycaprolactone (PCL) and gelatin (Gt) and displayed a thickness of 3-5 μm, with a high porosity and an average fiber diameter of 166 ± 85 nm. Human RPE cells seeded on the RWSF/PCL/Gt membranes showed a higher cell growth rate (p < 0.05), and a typical expression pattern of RPE signature genes, with reduced expression of inflammatory mediators. With long-term cultivation on the substrates, RPE cells exhibited characteristic polygonal morphology and development of apical microvilli. Immunocytochemisty demonstrated RPE-specific expression profiles in cells after 12-weeks of co-culture on RWSF/PCL/Gt membranes. Interestingly, the cells on the RWSF/PCL/Gt membranes functionally secreted polarized PEDF and phagocytosed labeled porcine POS. Furthermore, RWSF/PCL/Gt membranes transplanted subsclerally exhibited excellent biocompatibility without any evidence of inflammation or rejection. In conclusion, we established a novel RWSF-based substrate for growth of RPE cells with excellent cytocompatibility in vitro and biocompatibility in vivo for potential use as a prosthetic Bruch's membrane for RPE transplantation.

  8. Immunohistochemistry of the cytoskeleton of human prostatic epithelium. Evidence for disturbed organization in neoplasia.

    PubMed Central

    Purnell, D. M.; Heatfield, B. M.; Anthony, R. L.; Trump, B. F.

    1987-01-01

    An indirect immunoperoxidase technique was used to evaluate keratin, actin, tubulin, and calmodulin immunoreactivity in histologic sections of normal, hyperplastic, and neoplastic human prostate. Polyclonal as well as monoclonal keratin antibodies produced equivalent and intense staining of normal epithelium. The immunoreactivity of normal prostate with keratin antibodies was more pronounced than with antibodies to the other components of the cytoskeleton. Variation in staining for components of the cytoskeleton was minimal. The same findings applied to hyperplastic prostate. The immunoreactivity of prostate tumors with antibodies to these cytoskeletal proteins differed markedly from normal prostate. Prostatic carcinomas showed reduced keratin immunoreactivity with a panepithelial antibody, but unaltered or enhanced immunoreactivity with tubulin, actin, and calmodulin antibodies. Many tumors were unreactive with a monoclonal keratin antibody that was strongly reactive with tissues that contained cytokeratin 18 (45-kd) and which intensely stained normal and hyperplastic prostate. In addition, prostate carcinomas often yielded heterogeneous patterns of staining with actin, tubulin, and calmodulin antibodies in contrast to normal and hyperplastic prostate, which showed uniform staining. The results suggest that a disturbance in the organization of the cytoskeleton may accompany neoplastic transformation of human prostate. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2435158

  9. Activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium.

    PubMed

    Böttcher-Friebertshäuser, Eva; Klenk, Hans-Dieter; Garten, Wolfgang

    2013-11-01

    Influenza is an acute infection of the respiratory tract, which affects each year millions of people. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) through receptor binding and fusion of viral and endosomal membranes. HA is synthesized as a precursor protein and requires cleavage by host cell proteases to gain its fusion capacity. Although cleavage of HA is crucial for virus infectivity, little was known about relevant proteases in the human airways for a long time. Recent progress in the identification and characterization of HA-activating host cell proteases has been considerable however and supports the idea of targeting HA cleavage as a novel approach for influenza treatment. Interestingly, certain bacteria have been demonstrated to support HA activation either by secreting proteases that cleave HA or due to activation of cellular proteases and thereby may contribute to virus spread and enhanced pathogenicity. In this review, we give an overview on activation of influenza viruses by proteases from host cells and bacteria with the main focus on recent progress on HA cleavage by proteases HAT and TMPRSS2 in the human airway epithelium. In addition, we outline investigations of HA-activating proteases as potential drug targets for influenza treatment.

  10. Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.

    PubMed

    Milenkovic, Andrea; Brandl, Caroline; Milenkovic, Vladimir M; Jendryke, Thomas; Sirianant, Lalida; Wanitchakool, Potchanart; Zimmermann, Stephanie; Reiff, Charlotte M; Horling, Franziska; Schrewe, Heinrich; Schreiber, Rainer; Kunzelmann, Karl; Wetzel, Christian H; Weber, Bernhard H F

    2015-05-19

    In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.

  11. Aspects of nitrogen dioxide toxicity in environmental urban concentrations in human nasal epithelium

    SciTech Connect

    Koehler, C.; Ginzkey, C.; Friehs, G.; Hackenberg, S.; Froelich, K.; Scherzed, A.; Burghartz, M.; Kessler, M.; Kleinsasser, N.

    2010-06-01

    Cytotoxicity and genotoxicity of nitrogen dioxide (NO{sub 2}) as part of urban exhaust pollution are widely discussed as potential hazards to human health. This study focuses on toxic effects of NO{sub 2} in realistic environmental concentrations with respect to the current limit values in a human target tissue of volatile xenobiotics, the epithelium of the upper aerodigestive tract. Nasal epithelial cells of 10 patients were cultured as an air-liquid interface and exposed to 0.01 ppm NO{sub 2}, 0.1 ppm NO{sub 2}, 1 ppm NO{sub 2}, 10 ppm NO{sub 2} and synthetic air for half an hour. After exposure, genotoxicity was evaluated by the alkaline single-cell microgel electophoresis (Comet) assay and by induction of micronuclei in the micronucleus test. Depression of proliferation and cytotoxic effects were determined using the micronucleus assay and trypan blue exclusion assay, respectively. The experiments revealed genotoxic effects by DNA fragmentation starting at 0.01 ppm NO{sub 2} in the Comet assay, but no micronucleus inductions, no changes in proliferation, no signs of necrosis or apoptosis in the micronucleus assay, nor did the trypan blue exclusion assay show any changes in viability. The present data reveal a possible genotoxicity of NO{sub 2} in urban concentrations in a screening test. However, permanent DNA damage as indicated by the induction of micronuclei was not observed. Further research should elucidate the effects of prolonged exposure.

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

    PubMed Central

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

    2015-01-01

    AIM To investigate the cytotoxic effect on human corneal epithelial cells (HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose (CMC) and hyaluronic acid (HA). METHODS HCECs were exposed to 0.5% CMC (Refresh plus®, Allergan, Irvine, California, USA) and 0.1% and 0.3% HA (Kynex®, Alcon, Seoul, Korea, and Hyalein mini®, Santen, Osaka, Japan) for the period of 30min, and 4, 12, and 24h. Methyl thiazolyl tetrazolium (MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase (LDH) leakage assay to assess the cytotoxicity. Apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24h after confluent HCECs were scratch wounded. RESULTS The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells were demonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC. CONCLUSION CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds. PMID:25938030

  13. In situ localization of cytoskeletal elements in the human trabecular meshwork and cornea.

    PubMed

    Weinreb, R N; Ryder, M I

    1990-09-01

    The authors compared cytoskeletal elements of the in situ human trabecular-meshwork cell with in situ human corneal cells using indirect immunofluorescence staining for tubulin and intermediate filaments (vimentin, cytokeratin, and desmin) and NBD-phallacidin staining for f-actin using both fixed frozen and unfixed frozen sections from postmortem eyes. Both f-actin and tubulin were found throughout the cell body of trabecular-meshwork cells, keratocytes, corneal endothelium, and corneal epithelium. The f-actin staining pattern was concentrated at the cell periphery of these four cell types. Vimentin stain was intensely localized in focal areas of the trabecular-meshwork cell, keratocytes, and throughout the corneal endothelium. A general anticytokeratin antibody was intensely localized in corneal epithelium and endothelium. However, PKK-1 anticytokeratin antibody was seen only in superficial layers of corneal epithelium and not in corneal endothelium. The 4.62 anticytokeratin antibody was not observed in either corneal epithelium or endothelium. None of these three cytokeratin antibodies were seen in trabecular-meshwork cells or keratocytes. Desmin stain was not noted in any of these cell types. In general, cytoskeletal staining of unfixed frozen sections showed a similar staining pattern for f-actin and tubulin but a more uniform and intense staining pattern for vimentin and cytokeratin compared with fixed frozen material. The authors conclude that these cytoskeletal stains can differentiate human trabecular-meshwork cells from cells of the cornea in situ.

  14. A Biophysical Model for Integration of Electrical, Osmotic, and pH Regulation in the Human Bronchial Epithelium

    PubMed Central

    Falkenberg, Cibele V.; Jakobsson, Eric

    2010-01-01

    Abstract A dynamical biophysical model for the functioning of an epithelium is presented. This model integrates the electrical and osmotic behaviors of the epithelium, taking into account intracellular conditions. The specific tissue modeled is the human bronchial epithelium, which is of particular interest, as it is the location of the most common lethal symptoms of cystic fibrosis. The model is implemented in a modular form to facilitate future application of the code to other epithelial tissue by inputting different transporters, channels, and geometric parameters. The model includes pH regulation as an integral component of overall regulation of epithelial function, through the interdependence of pH, bicarbonate concentration, and current. The procedures for specification, the validation of the model, and parametric studies are presented using available experimental data of cultured human bronchial epithelium. Parametric studies are performed to elucidate a), the contribution of basolateral chloride channels to the short-circuit current functional form, and b), the role that regulation of basolateral potassium conductance plays in epithelial function. PMID:20409466

  15. Expression of intercellular adhesion molecule 1 (ICAM-1) on the human oviductal epithelium and mediation of lymphoid cell adherence.

    PubMed

    Utreras, E; Ossandon, P; Acuña-Castillo, C; Varela-Nallar, L; Müller, C; Arraztoa, J A; Cardenas, H; Imarai, M

    2000-09-01

    The epithelium of the human oviduct expresses the major histocompatibility complex (MHC) class II and shows endocytic properties towards luminal antigens. Therefore, the epithelial cells might behave as antigen-presenting cells, inducing a local immune response. The activation of antigen-specific T cells not only requires presentation of the peptide antigen by MHC class II, but also the presence of co-stimulatory molecules in the antigen-presenting cells. Therefore, the expression of the intercellular adhesion molecule 1 (ICAM-1) was examined in the epithelium of the human oviduct. Most oviducts showed epithelial ICAM-1 expression, as assessed by immunocytochemistry, western blot analysis and RT-PCR assay, and the expression was restricted to the luminal border of ciliated and secretory cells. Interferon gamma, interleukin 1 and lipopolysaccharide treatments increased the percentage of ICAM-1-positive cells in primary cultures, indicating that the expression of ICAM-1 in the oviduct might be upregulated in vivo by inflammatory cytokines or bacterial infections. Binding assays between allogenic phytohaemagglutinin-activated lymphocytes and epithelial monolayers expressing ICAM-1 demonstrated that this molecule stimulated lymphocyte adherence. The presence of ICAM-1, in addition to MHC class II, supports the putative role of the oviductal epithelium in antigen presentation. The exclusive apical distribution of ICAM-1 indicates that T-cell activation would occur in a polarized manner. Binding of lymphoid cells to the surface of the oviductal epithelium may help to retain these immune cells that are required for the clearance of pathogens.

  16. Corneal transplant - slideshow

    MedlinePlus

    ... ency/presentations/100082.htm Corneal transplant - series—Normal anatomy To use the sharing features on this page, ... Bethesda, MD 20894 U.S. Department of Health and Human Services National Institutes of Health Page last updated: ...

  17. Intestinal Trefoil Factor/TFF3 Promotes Re-epithelialization of Corneal Wounds*

    PubMed Central

    Paulsen, Friedrich P.; Woon, Chee-Wai; Varoga, Deike; Jansen, Anne; Garreis, Fabian; Jäger, Kristin; Amm, Marita; Podolsky, Daniel K.; Steven, Philipp; Barker, Nicholas P.; Sel, Saadettin

    2008-01-01

    Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3+/+) and Tff3-deficient (Tff3-/-) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3-/- mice is significantly prolonged in comparison to Tff3+/+ mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3+/+ and Tff3-/- mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds. PMID:18326859

  18. Intestinal trefoil factor/TFF3 promotes re-epithelialization of corneal wounds.

    PubMed

    Paulsen, Friedrich P; Woon, Chee-Wai; Varoga, Deike; Jansen, Anne; Garreis, Fabian; Jäger, Kristin; Amm, Marita; Podolsky, Daniel K; Steven, Philipp; Barker, Nicholas P; Sel, Saadettin

    2008-05-09

    Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3(+)(/)(+)) and Tff3-deficient (Tff3(-)(/)(-)) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3(-/-) mice is significantly prolonged in comparison to Tff3(+/+) mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3(+/+) and Tff3(-/-) mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.

  19. Gene expression of proteases and protease inhibitors in the human ciliary epithelium and ODM-2 cells.

    PubMed

    Ortego, J; Escribano, J; Coca-Prados, M

    1997-08-01

    Complementary DNAs (cDNAs), corresponding to the human proteinases cathepsins D and O and proteinase inhibitors alpha2-macroglobulin and PP5/TFPI-2, have recently been isolated and identified from a subtractive human ciliary body library. In the present study we determined: (i) their pattern of expression in the human eye; (ii) the ability of the ciliary body and/or ciliary epithelial cells to synthesize and secrete cathepsin D and alpha1-antitrypsin in vitro; and (iii) whether alpha1-antitrypsin expression in cultured ciliary epithelial cells is modulated by protein kinase C activation. Northern analysis demonstrated that the ciliary body expresses high levels of cathepsins D and O, alpha2-macroglobulin, alpha1-antitrypsin and PP5/TFPI-2 transcripts. Western blot analysis and immunoprecipitation experiments with cathepsin D and alpha1-antitrypsin antibodies indicated that metabolically labeled ciliary body explants and/or ciliary epithelial cells in vitro with 35S-methionine, synthesize and secrete these proteins. Cultured nonpigmented ciliary epithelial ODM-2 cells, in response to phorbol-12-myristate 13-acetate (PMA), but not to the non-protein kinase C binding phorbol ester 4 alpha-phorbol didecanoate (PDBu), elicited up-regulation (up to 5-fold) of transcription, synthesis and secretion of alpha1-antitrypsin. These results provide in vitro evidence that the ciliary epithelium synthesizes and secretes a selective group of proteinases and proteinase inhibitors detected also in aqueous humor. The expression of at least of one of the proteinase inhibitors, alpha1-antitrypsin, can be modulated in response to phorbol ester.

  20. Monocarboxylate transporter mediated uptake of moxifloxacin on human retinal pigmented epithelium cells

    PubMed Central

    Barot, Megha; Gokulgandhi, Mitan R.; Agrahari, Vibhuti; Pal, Dhananjay; Mitra, Ashim K.

    2015-01-01

    Objectives This work was aim to determine in vitro interaction of moxifloxacin with monocarboxylate transporter (MCT) using a human retinal pigment epithelium cells (ARPE-19). Methods In vitro moxifloxacin uptakes were performed at 37°C across ARPE-19 cells. Concentration-dependent uptake of moxifloxacin was performed to delineate moxifloxacin kinetics with MCT. Effects of MCT substrates, MCT inhibitors, pH and metabolic inhibitors on moxifloxacin uptake were conducted to delineate mechanism of moxifloxacin influx via MCT. Key findings Moxifloxacin uptake was found to exhibit saturable kinetics (Km = 1.56 ± 0.32 μM and Vmax = 0.58 ± 0.16 μM/min/mg protein). Higher uptake of moxifloxacin was observed at acidic pH. MCT substrates such as salisylic acid, ofloxacin and L-lactic acid significantly inhibited the uptake of moxifloxacin. Furthermore, moxifloxacin uptake was significantly reduced in the presence of metabolic and MCT inhibitors. Overall, this study demonstrated an interaction of moxifloxacin with Na+ and H+-coupled transporter, most likely MCT1. Conclusions Apart from the lipophilicity, we anticipate that lowest vitreal half-life of intravitreal moxifloxacin compared with other fluoroquinolones may be due to its interaction with MCT. This information might be crucial in clinical settings and can be further explored to improve vitreous half-life and therapeutic efficacy of moxifloxacin. PMID:24102496

  1. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells.

    PubMed

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-06-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases.

  2. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    NASA Astrophysics Data System (ADS)

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-06-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases.

  3. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    PubMed Central

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-01-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases. PMID:27246808

  4. A comparative study of candidal invasion in rabbit tongue mucosal explants and reconstituted human oral epithelium.

    PubMed

    Jayatilake, J A M S; Samaranayake, Y H; Samaranayake, L P

    2008-06-01

    The purpose of this study is to compare the light and scanning electron microscopic (SEM) features of tissue invasion by three Candida species (C. albicans, C. tropicalis, and C. dubliniensis) in two different tissue culture models: rabbit tongue mucosal explants (RTME) and reconstituted human oral epithelium (RHOE). Tongue mucosal biopsies of healthy New Zealand rabbits were maintained in explant culture using a transwell system. RHOE was obtained from Skinethic Laboratory (Nice, France). RTME and RHOE were inoculated with C. albicans, C. tropicalis, and C. dubliniensis separately and incubated at 37 degrees C, 5% CO(2), and 100% humidity up to 48 h. Light microscopic and SEM examinations of uninfected (controls) and infected tissues were performed at 24 and 48 h. C. albicans produced characteristic hallmarks of pathological tissue invasion in both tissue models over a period of 48 h. Hyphae penetrated through epithelial cells and intercellular gaps latter resembling thigmotropism. SEM showed cavitations on the epithelial cell surfaces particularly pronounced at sites of hyphal invasion. Some hyphae on RTME showed several clusters of blastospores attached in regular arrangements resembling "appareil sporifere". C. tropicalis and C. dubliniensis produced few hyphae mainly on RTME but they did not penetrate either model. Our findings indicate that multiple host-fungal interactions such as cavitations, thigmotropism, and morphogenesis take place during candidal tissue invasion. RTME described here appears to be useful in investigations of such pathogenic processes of Candida active at the epithelial front.

  5. Heat shock response and thermal resistance in cultured human retinal pigment epithelium.

    PubMed

    Wakakura, M; Foulds, W S

    1993-01-01

    The heat shock response was examined in cultured human retinal pigment epithelium (RPE) using indirect immunofluorescence. Mild head shock (39.5-40 degrees C for 1 hr) caused no changes in cell morphology and cells continued to produce the intermediate filament proteins, cytokeratin (keratin) and vimentin. In addition, cells subjected to mild heat shock demonstrated the presence of a heat shock protein (HSP-90). After severe heat shock (45.5-46 degrees C for 1 hr) most cells showed marked morphological changes and, in addition, HSP-90 and/or stress-induced 40 kDa protein production was significantly enhanced. The expression of vimentin was relatively well preserved whereas that of keratin was markedly reduced. When the more severe grade of heat shock was preceded by mild heat shock 20-24 hr earlier, the subsequent severe heat shock resulted in less marked morphological change than in cells not preconditioned and, in addition, the expression of both vimentin and keratin was relatively well preserved. Mildly heat shocked cells appeared to gain thermal resistance supporting the theory that the concomitant synthetic capacity for HSP and normal cellular proteins contributes to thermal resistance. In doubly heat shocked cells, however, HSP-90 expression was not enhanced. The discrepancy between the expression of HSP and thermal resistance is discussed.

  6. Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.

    PubMed

    Zhang, Pingbo; Kirby, David; Dufresne, Craig; Chen, Yan; Turner, Randi; Ferri, Sara; Edward, Deepak P; Van Eyk, Jennifer E; Semba, Richard D

    2016-04-01

    The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of <0.1% and <1%, respectively. Forty-three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four "missing proteins" were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.

  7. Expression of Human β-Defensins in Conjunctival Epithelium: Relevance to Dry Eye Disease

    PubMed Central

    Narayanan, Srihari; Miller, William L.; McDermott, Alison M.

    2006-01-01

    Purpose. The goals of this study were to investigate whether β-defensins are differentially expressed in the conjunctival epithelium of patients with moderate dry eye when compared with normal subjects and whether proinflammatory cytokines or bacteria can modulate the expression of human β-defensins (hBDs)-1, -2, and -3 by conjunctival epithelial cells. Methods. RNA extracted from conjunctival impression cytology specimens of eight normal subjects and nine patients with moderate dry eye was used in RT-PCR to detect mRNA for hBDs-1, -2, and -3. Two conjunctival epithelial cell lines and primary cultured conjunctival epithelial cells were treated with proinflammatory cytokines or heat-killed Pseudomonas aeruginosa. RT-PCR and immunoblot analysis were used to detect mRNA for hBD-1, -2, and -3 and protein secretion of hBD-2, respectively. Results. hBD-2 message was detected in RNA samples of eight of nine patients with dry eye, but not in any of the normal subjects’ samples, whereas hBD-1 and -3 were detected in all subjects tested. RT-PCR revealed an upregulation of hBD-2 but no difference in expression of hBD-1 and -3 in cultured conjunctival cells after a 24-hour treatment with 10 ng/mL interleukin (IL)-1β, IL-1β and tumor necrosis factor-α (10 ng/mL) or heat-killed Pseudomonas aeruginosa (1 million colony-forming units; n = 3). hBD-2 expression was upregulated from 4 hours of treatment with IL-1β (at 10 ng/mL; (n = 2–3) and at a concentration of 0.1 ng/mL IL-1β (24-hour treatment; n = 2–3). Immunoblots demonstrated protein secretion results corresponding to the RT-PCR data. Conclusions. hBD-2 was expressed only in the conjunctival epithelium of patients with moderate dry eye. Because cytokines such as IL-1β and TNF-α induced the expression of hBD-2 by conjunctival epithelial cells and because increased proinflammatory cytokine activity is a feature of dry eye disease, it can be speculated that the hBD-2 upregulation observed in subjects with moderate

  8. POU2AF1 Functions in the Human Airway Epithelium To Regulate Expression of Host Defense Genes.

    PubMed

    Zhou, Haixia; Brekman, Angelika; Zuo, Wu-Lin; Ou, Xuemei; Shaykhiev, Renat; Agosto-Perez, Francisco J; Wang, Rui; Walters, Matthew S; Salit, Jacqueline; Strulovici-Barel, Yael; Staudt, Michelle R; Kaner, Robert J; Mezey, Jason G; Crystal, Ronald G; Wang, Guoqing

    2016-04-01

    In the process of seeking novel lung host defense regulators by analyzing genome-wide RNA sequence data from normal human airway epithelium, we detected expression of POU domain class 2-associating factor 1 (POU2AF1), a known transcription cofactor previously thought to be expressed only in lymphocytes. Lymphocyte contamination of human airway epithelial samples obtained by bronchoscopy and brushing was excluded by immunohistochemistry staining, the observation of upregulation of POU2AF1 in purified airway basal stem/progenitor cells undergoing differentiation, and analysis of differentiating single basal cell clones. Lentivirus-mediated upregulation of POU2AF1 in airway basal cells induced upregulation of host defense genes, including MX1, IFIT3, IFITM, and known POU2AF1 downstream genes HLA-DRA, ID2, ID3, IL6, and BCL6. Interestingly, expression of these genes paralleled changes of POU2AF1 expression during airway epithelium differentiation in vitro, suggesting POU2AF1 helps to maintain a host defense tone even in pathogen-free condition. Cigarette smoke, a known risk factor for airway infection, suppressed POU2AF1 expression both in vivo in humans and in vitro in human airway epithelial cultures, accompanied by deregulation of POU2AF1 downstream genes. Finally, enhancing POU2AF1 expression in human airway epithelium attenuated the suppression of host defense genes by smoking. Together, these findings suggest a novel function of POU2AF1 as a potential regulator of host defense genes in the human airway epithelium.

  9. Decreased expression of intelectin 1 in the human airway epithelium of smokers compared to nonsmokers.

    PubMed

    Carolan, Brendan J; Harvey, Ben-Gary; De, Bishnu P; Vanni, Holly; Crystal, Ronald G

    2008-10-15

    Lectins are innate immune defense proteins that recognize bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with an increased risk of infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey the expression of lectin genes in large airway epithelium from nine nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change; p < 0.05) in lectin gene expression among healthy smokers compared with nonsmokers except for down-regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in bacterial cell walls (large airway epithelium, p < 0.01; small airway epithelium, p < 0.01). This was confirmed by TaqMan RT-PCR in both large (p < 0.05) and small airway epithelium (p < 0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared with healthy nonsmokers (p < 0.02). Finally, compared with healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema and normal spirometry (n = 13, p < 0.01) and smokers with established chronic obstructive pulmonary disease (n = 14, p < 0.01). In the context that intelectin 1 plays a role in defense against bacteria, its down-regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers.

  10. Decreased Expression of Intelectin 1 in the Human Airway Epithelium of Smokers Compared to Nonsmokers*

    PubMed Central

    Carolan, Brendan J.; Harvey, Ben-Gary; De, Bishnu P.; Vanni, Holly; Crystal, Ronald G.

    2009-01-01

    Summary Lectins are innate immune defense proteins that recognize specific bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with increased risk of bacterial infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey expression of lectin genes in large airway epithelium from 9 nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change, p<0.05) in lectin gene expression among healthy smokers compared to nonsmokers except for a striking down regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in the cell walls of bacteria (large airway epithelium, p<0.01; small airway epithelium, p<0.01). This was confirmed by TaqMan RT-PCR in both large (p<0.05) and small airway epithelium (p<0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared to healthy nonsmokers (p<0.02). Finally, compared to healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema with normal spirometry (n= 13, p<0.01) and smokers with established COPD (n= 14, p<0.01). In the context that intelectin 1 is an epithelial molecule that likely plays a role in defense against bacteria, its down regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers, including those with COPD. PMID:18832735

  11. Stem Cell Derived Retinal Pigment Epithelium: The Role of Pigmentation as Maturation Marker and Gene Expression Profile Comparison with Human Endogenous Retinal Pigment Epithelium.

    PubMed

    Bennis, A; Jacobs, J G; Catsburg, L A E; Ten Brink, J B; Koster, C; Schlingemann, R O; van Meurs, J; Gorgels, T G M F; Moerland, P D; Heine, V M; Bergen, A A

    2017-07-21

    In age-related macular degeneration (AMD) the retinal pigment epithelium (RPE) deteriorates, leading to photoreceptor decay and severe vision loss. New therapeutic strategies aim at RPE replacement by transplantation of pluripotent stem cell (PSC)-derived RPE. Several protocols to generate RPE have been developed where appearance of pigmentation is commonly used as indicator of RPE differentiation and maturation. It is, however, unclear how different pigmentation stages reflect developmental stages and functionality of PSC-derived RPE cells. We generated human embryonic stem cell-derived RPE (hESC-RPE) cells and investigated their gene expression profiles at early pigmentation (EP) and late pigmentation (LP) stages. In addition, we compared the hESC-RPE samples with human endogenous RPE. We used a common reference design microarray (44 K). Our analysis showed that maturing hESC-RPE, upon acquiring pigmentation, expresses markers specific for human RPE. Interestingly, our analysis revealed that EP and LP hESC-RPE do not differ much in gene expression. Our data further showed that pigmented hESC-RPE has a significant lower expression than human endogenous RPE in the visual cycle and oxidative stress pathways. In contrast, we observed a significantly higher expression of pathways related to the process adhesion-to-polarity model that is typical of developing epithelial cells. We conclude that, in vitro, the first appearance of pigmentation hallmarks differentiated RPE. However, further increase in pigmentation does not result in much significant gene expression changes and does not add important RPE functionalities. Consequently, our results suggest that the time span for obtaining differentiated hESC-RPE cells, that are suitable for transplantation, may be greatly reduced.

  12. Cysteamine suppresses human peripheral blood mononuclear cells – human corneal endothelial cell reaction via reactive oxygen species reduction

    PubMed Central

    Hyon, Joon Young; Kim, Seonhowa; Koh, Jae Woong; Kwon, Soon Il; Wee, Won Ryang

    2011-01-01

    Purpose To investigate the effect of cysteamine (CYS) on mixed peripheral blood mononuclear cells (PBMCs) - human corneal endothelial cell (HCEC) reaction (MLER). Methods PBMC stimulation assay was performed using cultured HCEC. MLERs were treated with various concentrations of CYS (0–20 mM). The proliferation rate and secretion profiles of transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) of PBMCs stimulated by cultured HCEC were determined using bromodeoxyuridine proliferation assay and enzyme-linked immunosorbent assay, respectively. Results CYS suppressed PBMC proliferation in a dose-dependent manner (p<0.001). The intracellular reactive oxygen species (ROS) levels decreased with an increase in CYS concentration (p<0.001). The levels of TGF-β1 and IL-6 decreased in a dose-dependent manner as well (p=0.011 and 0.003, respectively). Conclusions This study showed that CYS decreased PBMC proliferation, IL-6 and TGF-β1 levels via ROS formation. Our results suggest that CYS could suppress inflammation associated with PBMCs to corneal endothelial cells. PMID:22219632

  13. Regulation of estrogen receptors and MMP-2 expression by estrogens in human retinal pigment epithelium.

    PubMed

    Marin-Castaño, Maria E; Elliot, Sharon J; Potier, Mylen; Karl, Michael; Striker, Liliane J; Striker, Gary E; Csaky, Karl G; Cousins, Scott W

    2003-01-01

    Age-related macular degeneration (ARMD) is characterized by progressive thickening and accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE). ECM dysregulation probably contributes to the pathologic course of ARMD. By activating estrogen receptors (ERs), estrogens regulate the expression of genes relevant in the turnover of ECM, among them matrix metalloproteinase (MMP)-2. Estrogen deficiency may predispose to dysregulated synthesis and degradation of ECM, leading to accumulation of collagens and other proteins between the RPE and its basement membrane. The purposes in the current study were to confirm the expression of ERs in human RPE, to elucidate whether these ERs are functional, and to test whether 17beta-estradiol (E(2)) regulates expression of ERs and MMP-2. Expression of ERs was examined in freshly isolated human RPE monolayer and in cultured human RPE cells, by using total RNA for RT-PCR and protein extracts for Western blot analysis. Supernatants were collected from freshly isolated human RPE and from cultured human RPE to assess MMP-2 activity by zymography and protein expression by Western blot. The transcriptional activity of ERs was studied in transfection experiments with an estrogen-responsive reporter construct. All these studies were preformed in the presence or absence of E(2) (10(-11) and 10(-7) M). Human RPE isolated from female and male individuals expressed both ER subtypes alpha and beta at the mRNA and protein levels. Treatment of cultured RPE cells with 10(-10) M E(2) increased expression of mRNA and protein of both receptor subtypes. E(2) (10(-10) M) also increased MMP-2 activity (approximately 2.2-fold) and protein expression (approximately 2.5-fold). In contrast, there was no change in ER levels and MMP-2 activity at higher E(2) concentrations (10(-8) M), compared with baseline. Preincubation of cells with 10(-7) M pyrrolidinedithiocarbamate (PDTC), an inhibitor of nuclear

  14. Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

    PubMed

    Hannan, Nicholas R F; Sampaziotis, Fotios; Segeritz, Charis-Patricia; Hanley, Neil A; Vallier, Ludovic

    2015-07-15

    Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development.

  15. Peptidoglycan Induces the Production of Interleukin-8 via Calcium Signaling in Human Gingival Epithelium

    PubMed Central

    Son, Aran; Hong, Jeong Hee

    2015-01-01

    The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells. PMID:25605997

  16. Unravelling the interplay of sphingolipids and TGF-β signaling in the human corneal stroma.

    PubMed

    Nicholas, Sarah E; Rowsey, Tyler G; Priyadarsini, Shrestha; Mandal, Nawajes A; Karamichos, Dimitrios

    2017-01-01

    To delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-β) in order to develop novel, non-invasive therapies. Human corneal fibroblasts (HCFs) were harvested from healthy donors, stimulated with Vitamin C to promote extracellular matrix assembly, treated with exogenous sphingosine-1-phosphate (S1P) or sphingosine kinase inhibitor 2 (SPHK I2) and isolated after 4 weeks for further analysis. Data showed that S1P led to a significant decrease in cellular migration where SPHK I2 just delayed it for 24h. Significant modulation of the sphingolipid pathway was also noted. Sphingosine kinase-1 (SphK1) was significantly downregulated upon exogenous stimulation with S1P at a concentration of 5μM and Sphingosine kinase-2 (SphK2) was also significantly downregulated at concentrations of 0.01μM, 0.1μM, and 5μM; whereas no effects were observed upon stimulation with SPHK I2. S1PR3 was significantly downregulated by 0.1μM and 5μM S1P and upregulated by 5μM and 10μM SPHK I2. Furthermore, both S1P and SPHK I2 regulated corneal fibrosis markers such as alpha-smooth muscle actin, collagen I, III, and V. We also investigated the interplay between two TGF-β isoforms and S1P/SPHK I2 treatments and found that TGF-β1 and TGF-β3 were both significantly upregulated with the 0.1μM S1P but were significantly downregulated with the 5μM S1P concentration. When TGF-β1 was compared directly to TGF-β3 expression, we observed that TGF-β3 was significantly downregulated compared to TGF-β1 in the 5μM concentration of S1P. No changes were observed upon SPHK I2 treatment. Our study delineates the role of sphingolipids in the human cornea and highlights their different activities based on the cell/tissue type.

  17. Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

    PubMed

    Feizi, Sepehr; Soheili, Zahra-Soheila; Bagheri, Abouzar; Balagholi, Sahar; Mohammadian, Azam; Rezaei-Kanavi, Mozhgan; Ahmadieh, Hamid; Samiei, Shahram; Negahban, Kambiz

    2014-05-01

    The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Immunohistochemical and immunoelectron microscopic analyses of alpha-amylase isozymes in human intrahepatic biliary epithelium and hepatocytes.

    PubMed

    Terada, T; Kono, N; Nakanuma, Y

    1992-11-01

    The expression and localization of the pancreatic and salivary isozymes of alpha-amylase in the intrahepatic biliary epithelium and hepatocytes were examined by the immunohistochemical method with polyclonal and monoclonal antibodies in 45 normal autopsied human livers. Immunoelectron microscopic studies with the protein A-gold method were performed with the monoclonal antibodies (MAb) on seven of the livers. The intrahepatic biliary system was divided into large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands. Immunohistochemically, pancreatic isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands in almost all livers. Interlobular ducts expressed pancreatic isozyme in only four (9%) livers. Bile ductules and hepatocytes were negative for pancreatic isozyme in all cases. Expression of salivary isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands in almost all livers, although the expression in interlobular ducts and bile ductules was weak. Hepatocytes were weakly positive for salivary isozyme. Immunoelectron microscopy revealed that both pancreatic and salivary isozymes were located in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands, and that hepatocytes had no pancreatic isozyme but contained salivary isozyme. These data suggest that pancreatic and salivary isozymes of alpha-amylase are produced by the intrahepatic biliary epithelium and secreted into intrahepatic biliary lumens, and that they may play an important role in the physiology of the intrahepatic biliary tree and hepatic bile. It is also suggested that hepatocytes produce a small amount of salivary alpha-amylase that may be secreted into the biliary tree.

  19. Content of trans fatty acids in human cheek epithelium: comparison with serum and adipose tissue.

    PubMed

    Abraham, Ransi A; Bahl, Vinay K; Parshad, Rajinder; Seenu, V; Roy, Ambuj; Golandaz, Smita; Dorairaj, Prabhakaran; Ramakrishnan, Lakshmy

    2013-01-01

    Studies pertaining to trans fatty acids (TFA), which have been implicated in development of chronic diseases, are more relevant in developing countries where nutrition transition is changing traditional habits and practices. Measuring TFA is an arduous task because of the need for fat biopsies. This study identifies a tissue, which can be easily accessed for analytical measurement of trans fatty acid. In this cross-sectional study, fatty acid in adipose tissue, cheek epithelium, and blood samples were assessed by gas chromatography. Spearman correlation coefficient was computed to study the correlation of fatty acid distribution among the three tissues. The correlation coefficient of total trans fatty acid between cheek epithelium and serum was 0.30 (P < 0.02) and between cheek epithelium and adipose tissue was 0.33 (P < 0.019). This study is the first to report trans fatty acid profile in cheek epithelium giving scope for utilizing the cheek epithelium as a tissue for objective assessment of trans fatty acid intake.

  20. Effects of proinflammatory cytokines on the claudin-19 rich tight junctions of human retinal pigment epithelium.

    PubMed

    Peng, Shaomin; Gan, Geliang; Rao, Veena S; Adelman, Ron A; Rizzolo, Lawrence J

    2012-07-27

    Chronic, subclinical inflammation contributes to the pathogenesis of several ocular diseases, including age-related macular degeneration. Proinflammatory cytokines affect tight junctions in epithelia that lack claudin-19, but in the retinal pigment epithelium claudin-19 predominates. We examined the effects of cytokines on the tight junctions of human fetal RPE (hfRPE). hfRPE was incubated with interleukin 1-beta (IL-1β), interferon-gamma (IFNγ), or tumor necrosis factor-alpha (TNFα), alone or in combination. Permeability and selectivity of the tight junctions were assessed using nonionic tracers and electrophysiology. Claudins, occludin, and ZO-1 were examined using PCR, immunoblotting, and confocal immunofluorescence microscopy. Only TNFα consistently reduced transepithelial electrical resistance (TER) >80%. A serum-free medium revealed two effects of TNFα: (1) decreased TER was observed only when TNFα was added to the apical side of the monolayer, and (2) expression of TNFα receptors and inhibitors of apoptosis were induced from either side of the monolayer. In untreated cultures, tight junctions were slightly cation selective, and this was affected minimally by TNFα. The results were unexplained by effects on claudin-2, claudin-3, claudin-19, occludin, and ZO-1, but changes in the morphology of the junctions and actin cytoskeleton may have a role. Claudin-19-rich tight junctions have low permeability for ionic and nonionic solutes, and are slightly cation-selective. Claudin-19 is not a direct target of TNFα. TNFα may protect RPE from apoptosis, but makes the monolayer leaky when it is presented to the apical side of the monolayer. Unlike other epithelia, IFNγ failed to augment the effect of TNFα on tight junctions.

  1. Effects of Proinflammatory Cytokines on the Claudin-19 Rich Tight Junctions of Human Retinal Pigment Epithelium

    PubMed Central

    Peng, Shaomin; Gan, Geliang; Rao, Veena S.; Adelman, Ron A.; Rizzolo, Lawrence J.

    2012-01-01

    Purpose. Chronic, subclinical inflammation contributes to the pathogenesis of several ocular diseases, including age-related macular degeneration. Proinflammatory cytokines affect tight junctions in epithelia that lack claudin-19, but in the retinal pigment epithelium claudin-19 predominates. We examined the effects of cytokines on the tight junctions of human fetal RPE (hfRPE). Methods. hfRPE was incubated with interleukin 1-beta (IL-1β), interferon-gamma (IFNγ), or tumor necrosis factor-alpha (TNFα), alone or in combination. Permeability and selectivity of the tight junctions were assessed using nonionic tracers and electrophysiology. Claudins, occludin, and ZO-1 were examined using PCR, immunoblotting, and confocal immunofluorescence microscopy. Results. Only TNFα consistently reduced transepithelial electrical resistance (TER) >80%. A serum-free medium revealed two effects of TNFα: (1) decreased TER was observed only when TNFα was added to the apical side of the monolayer, and (2) expression of TNFα receptors and inhibitors of apoptosis were induced from either side of the monolayer. In untreated cultures, tight junctions were slightly cation selective, and this was affected minimally by TNFα. The results were unexplained by effects on claudin-2, claudin-3, claudin-19, occludin, and ZO-1, but changes in the morphology of the junctions and actin cytoskeleton may have a role. Conclusions. Claudin-19–rich tight junctions have low permeability for ionic and nonionic solutes, and are slightly cation-selective. Claudin-19 is not a direct target of TNFα. TNFα may protect RPE from apoptosis, but makes the monolayer leaky when it is presented to the apical side of the monolayer. Unlike other epithelia, IFNγ failed to augment the effect of TNFα on tight junctions. PMID:22761260

  2. Quantitative Autofluorescence and Cell Density Maps of the Human Retinal Pigment Epithelium

    PubMed Central

    Ach, Thomas; Huisingh, Carrie; McGwin, Gerald; Messinger, Jeffrey D.; Zhang, Tianjiao; Bentley, Mark J.; Gutierrez, Danielle B.; Ablonczy, Zsolt; Smith, R. Theodore; Sloan, Kenneth R.; Curcio, Christine A.

    2014-01-01

    Purpose. Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF. Methods. Retinal pigment epithelium–Bruch's membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.2 hours; no retinal pathologies), preserving foveal position. Phalloidin-binding RPE cytoskeleton and LF-AF (488-nm excitation) were imaged at up to 90 predefined positions. Maps were assembled from 83,330 cells in 1470 locations. From Voronoi regions representing each cell, the number of neighbors, cell area, and total AF intensity normalized to an AF standard was determined. Results. Highly variable between individuals, RPE-AF increases significantly with age. A perifoveal ring of high AF mirrors rod photoreceptor topography and fundus-AF. Retinal pigment epithelium cell density peaks at the fovea, independent of age, yet no net RPE cell loss is detectable. The RPE monolayer undergoes considerable lifelong re-modeling. The relationship of cell size and AF, a surrogate for LF concentration, is orderly and linear in both groups. Autofluorescence topography differs distinctly from the topography of age-related rod loss. Conclusions. Digital maps of quantitative AF, cell density, and packing geometry provide metrics for cellular-resolution clinical imaging and model systems. The uncoupling of RPE LF content, cell number, and photoreceptor topography in aging challenges LF's role in AMD. PMID:25034602

  3. Prevalence of human papillomavirus in the cervical epithelium of Mexican women: meta-analysis

    PubMed Central

    2012-01-01

    Background Human Papillomavirus (HPV) in cervical epithelium has been identified as the main etiological factor in the developing of Cervical Cancer (CC), which has recently become a public health problem in Mexico. This finding has allowed for the development of vaccines that help prevent this infection. In the present study, we aimed to determine the prevalence and HPV type-distribution in Mexican women with CC, high-grade squamous intraepithelial lesion (HSIL), low-grade squamous intraepithelial lesion (LSIL), and Normal cytology (N) to estimate the impact of the HPV vaccines. Methods The PubMed database was used to identify and review all articles that reported data on HPV prevalence in CC, precursor lesions, and normal cytology of Mexican women. Results A total of 8,706 samples of the tissues of Mexican women were stratified according to diagnosis as follows: 499 for CC; 364 for HSIL; 1,425 for LSIL, and 6,418 for N. According to the results, the most prevalent genotypes are the following: HPV16 (63.1%), -18 (8.6%), -58, and −31 (5%) for CC; HPV-16 (28.3%), 58 (12.6%), 18 (7.4%), and 33 (6.5%) for HSIL; HPV-16 (13.1%), 33 (7.4%), 18 (4.2%), and 58 (2.6%) for LSIL, and HPV-16 (3.4%), 33 (2.1%), 18, and 58 (1.2%) for N. Conclusions Taken together, genotypes 58 and 31 (10%) are more common than type 18 (8.6%) in CC. Therefore, the inclusion of these two genotypes in a second-generation vaccine would provide optimal prevention of CC in Mexico. PMID:23199368

  4. Correspondence regarding "Effect of active smoking on the human bronchial epithelium transcriptome"

    PubMed Central

    Zuyderduyn, Scott D

    2009-01-01

    Background In the work of Chari et al. entitled "Effect of active smoking on the human bronchial epithelium transcriptome" the authors use SAGE to identify candidate gene expression changes in bronchial brushings from never, former, and current smokers. These gene expression changes are categorized into those that are reversible or irreversible upon smoking cessation. A subset of these identified genes is validated on an independent cohort using RT-PCR. The authors conclude that their results support the notion of gene expression changes in the lungs of smokers which persist even after an individual has quit. Results This correspondence raises questions about the validity of the approach used by the authors to analyze their data. The majority of the reported results suffer deficiencies due to the methods used. The most fundamental of these are explained in detail: biases introduced during data processing, lack of correction for multiple testing, and an incorrect use of clustering for gene discovery. A randomly generated "null" dataset is used to show the consequences of these shortcomings. Conclusion Most of Chari et al.'s findings are consistent with what would be expected by chance alone. Although there is clear evidence of reversible changes in gene expression, the majority of those identified appear to be false positives. However, contrary to the authors' claims, no irreversible changes were identified. There is a broad consensus that genetic change due to smoking persists once an individual has quit smoking; unfortunately, this study lacks sufficient scientific rigour to support or refute this hypothesis or identify any specific candidate genes. The pitfalls of large-scale analysis, as exemplified here, may not be unique to Chari et al. PMID:19224643

  5. Imaging human retinal pigment epithelium cells using adaptive optics optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Liu, Zhuolin; Kocaoglu, Omer P.; Turner, Timothy L.; Miller, Donald T.

    2016-03-01

    Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, but are often compromised in ageing and major ocular diseases that lead to blindness. Early manifestation of RPE disruption occurs at the cellular level, and while biomarkers at this scale hold considerable promise, RPE cells have proven extremely challenging to image in the living human eye. We present a novel method based on optical coherence tomography (OCT) equipped with adaptive optics (AO) that overcomes the associated technical obstacles. The method takes advantage of the 3D resolution of AO-OCT, but more critically sub-cellular segmentation and registration that permit organelle motility to be used as a novel contrast mechanism. With this method, we successfully visualized RPE cells and characterized their 3D reflectance profile in every subject and retinal location (3° and 7° temporal to the fovea) imaged to date. We have quantified RPE packing geometry in terms of cell density, cone-to-RPE ratio, and number of nearest neighbors using Voronoi and power spectra analyses. RPE cell density (cells/mm2) showed no significant difference between 3° (4,892+/-691) and 7° (4,780+/-354). In contrast, cone-to- RPE ratio was significantly higher at 3° (3.88+/-0.52:1) than 7° (2.31+/- 0.23:1). Voronoi analysis also showed most RPE cells have six nearest neighbors, which was significantly larger than the next two most prevalent associations: five and seven. Averaged across the five subjects, prevalence of cells with six neighbors was 51.4+/-3.58% at 3°, and 54.58+/-3.01% at 7°. These results are consistent with histology and in vivo studies using other imaging modalities.

  6. SLURP-2: A novel cholinergic signaling peptide in human mucocutaneous epithelium.

    PubMed

    Arredondo, Juan; Chernyavsky, Alexander I; Jolkovsky, David L; Webber, Robert J; Grando, Sergei A

    2006-07-01

    The biologic role of novel cholinergic toxin-like signaling peptides termed SLURP (secreted mammalian Ly-6/uPAR-related protein) in the mucocutaneous epithelium is a subject of intense research. Previous studies demonstrated that SLURP-1 activates the alpha7 subtype of keratinocyte nicotinic acetylcholine receptors (nAChRs) and facilitates keratinization and programmed cell death, and that the level of SLURP-2 was found to be upregulated several fold in the hyperproliferative skin of patients with psoriasis. In this study, we demonstrated for the first time that human epidermal and oral keratinocytes secrete SLURP-2. We cloned human SLURP-2 and produced the mouse monoclonal antibody 341F10-1F12 that visualized SLURP-2 in the cytoplasm of normal human epidermal and oral keratinocytes grown in culture. In epidermis, SLURP-2 was found predominantly in the suprabasal compartment, whereas in the attached gingiva-in the lowermost epithelial layers. Recombinant SLURP-2 (rSLURP-2) competed with nicotinic radioligands for binding to keratinocytes, showing a higher affinity to the [3H]epibatidine- than [3H]nicotine-labeled sites. Treatment with rSLURP-2 significantly (P < 0.05) increased the number of keratinocytes in culture and their resistance to apoptosis, which could be abolished by mecamylamine more efficiently than alpha-bungarotoxin. By real-time PCR and in-cell western, rSLURP-2 significantly (P < 0.05) downregulated gene expression of the differentiation markers loricrin, filaggrin, and cytokeratins 1 and 10, and pro-apoptotic Bax, Bad, and caspase 3 which were elevated by high extracellular calcium, and rSLURP-2 also abolished activation of caspases 3 and 8 caused by camptothecin. These results indicated that SLURP-2 competes with acetylcholine predominantly at the alpha3 nAChR, and that receptor ligation with SLURP-2 delays keratinocyte differentiation and prevents apoptosis. Thus, the different effects observed for SLURP-1 and -2 can be explained by their

  7. Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium

    PubMed Central

    Kinnick, Tyson R.; Stanton, J. Brett; Johnson, Adiv A.; Lynch, Ronald M.; Marmorstein, Lihua Y.

    2015-01-01

    Purpose Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1. Methods Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp. Results Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control

  8. Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization

    PubMed Central

    Han, Kyu-Yeon; Tran, Jennifer A.; Chang, Jin-Hong; Azar, Dimitri T.; Zieske, James D.

    2017-01-01

    Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions. PMID:28165027

  9. Rhinovirus Delays Cell Repolarization in a Model of Injured/Regenerating Human Airway Epithelium

    PubMed Central

    Faris, Andrea N.; Ganesan, Shyamala; Chattoraj, Asamanja; Chattoraj, Sangbrita S.; Comstock, Adam T.; Unger, Benjamin L.; Hershenson, Marc B.

    2016-01-01

    Rhinovirus (RV), which causes exacerbation in patients with chronic airway diseases, readily infects injured airway epithelium and has been reported to delay wound closure. In this study, we examined the effects of RV on cell repolarization and differentiation in a model of injured/regenerating airway epithelium (polarized, undifferentiated cells). RV causes only a transient barrier disruption in a model of normal (mucociliary-differentiated) airway epithelium. However, in the injury/regeneration model, RV prolongs barrier dysfunction and alters the differentiation of cells. The prolonged barrier dysfunction caused by RV was not a result of excessive cell death but was instead associated with epithelial-to-mesenchymal transition (EMT)-like features, such as reduced expression of the apicolateral junction and polarity complex proteins, E-cadherin, occludin, ZO-1, claudins 1 and 4, and Crumbs3 and increased expression of vimentin, a mesenchymal cell marker. The expression of Snail, a transcriptional repressor of tight and adherence junctions, was also up-regulated in RV-infected injured/regenerating airway epithelium, and inhibition of Snail reversed RV-induced EMT-like features. In addition, compared with sham-infected cells, the RV-infected injured/regenerating airway epithelium showed more goblet cells and fewer ciliated cells. Inhibition of epithelial growth factor receptor promoted repolarization of cells by inhibiting Snail and enhancing expression of E-cadherin, occludin, and Crumbs3 proteins, reduced the number of goblet cells, and increased the number of ciliated cells. Together, these results suggest that RV not only disrupts barrier function, but also interferes with normal renewal of injured/regenerating airway epithelium by inducing EMT-like features and subsequent goblet cell hyperplasia. PMID:27119973

  10. [Regeneration and fibrosis of corneal tissues].

    PubMed

    Simirskiĭ, V N

    2014-01-01

    In this review, the features of the regeneration of corneal tissue and its disorders leading to the development of fibrosis are considered. The data on the presence of stem (clonogenic) cell pool in the corneal tissues (epithelium, endothelium, stroma) are given; these cells can serve as a source for regeneration of the tissues at injury or various diseases. The main steps of regeneration of corneal tissues and their disorders that lead to outstripping proliferation of myofibroblasts and secretion of extracellular matrix in the wound area and eventually cause the formation of connective tissue scar and corneal opacity are considered. Particular attention is given to the successes of translational medicine in the treatment of corneal tissue fibrosis. The methods of cell therapy aimed at the restoration of stem cell pool of corneal tissues are the most promising. Gene therapy provides more opportunities; one of its main objectives is the suppression of the myofibroblast proliferation responsible for the development of fibrosis.

  11. Sub-micrometer axial resolution OCT for in-vivo imaging of the cellular structure of healthy and keratoconic human corneas

    PubMed Central

    Bizheva, Kostadinka; Tan, Bingyao; MacLelan, Benjamin; Kralj, Olivera; Hajialamdari, Mojtaba; Hileeto, Denise; Sorbara, Luigina

    2017-01-01

    Corneal degenerative conditions such as keratoconus (KC) cause progressive damage to the anterior corneal tissue and eventually severely compromise visual acuity. The ability to visualize corneal tissue damage in-vivo at cellular or sub-cellular level at different stages of development of KC and other corneal diseases, can aid the early diagnostics as well as the development of more effective treatment approaches for various corneal pathologies, including keratoconus. Here, we present the optical design of an optical coherence tomography system that can achieve 0.95 µm axial resolution in biological tissue and provide test results for the system’s spatial resolution and sensitivity. Corneal images acquired in-vivo with this system from healthy and keratoconic human subjects reveal the cellular and sub-cellular structure of the corneal epithelium, as well as the normal and abnormal structure of the Bowman’s membrane and the anterior corneal stroma. PMID:28270986

  12. Long‐term recovery of the human corneal endothelium after toxic injury by benzalkonium chloride

    PubMed Central

    Hughes, E H; Pretorius, M; Eleftheriadis, H; Liu, C S C

    2007-01-01

    Introduction The inadvertent intra‐ocular administration of benzalkonium chloride‐preserved hydroxypropyl methylcellulose during cataract surgery at another hospital in 1999 resulted in toxic corneal endothelial injury and profound postoperative corneal oedema as a result of endothelial decompensation. The long‐term effect of this adverse event was assessed. Methods All 19 patients were invited to return for examination including corneal endothelial specular microscopy and pachymetry seven years after the incident. Results were compared with data from one year after the incident. Results Five patients attended for examination, one had received a penetrating keratoplasty and was, therefore, excluded. Ten patients had died and four had moved out of the region and were unable to attend. All four study patients were pain free and achieved 6/12 or better. Mean central corneal thickness reduced by 13% from 652.6 μm at one year to 563.4 μm. Mean central corneal endothelial cell density (n  =  3) increased 28% from 663.7 cells/mm2 at one year to 835.7 cells/mm2 (p<0.05). Conclusions After toxic injury, corneal endothelial function may have a remarkable capacity for recovery even after the first postoperative year. The rise in central endothelial cell density may represent cell migration from less affected areas or cellular proliferation. Should this unfortunate event recur, clinicians may expect continued recovery beyond one year. PMID:17504856

  13. Corneal biomechanical properties from air-puff corneal deformation imaging

    NASA Astrophysics Data System (ADS)

    Marcos, Susana; Kling, Sabine; Bekesi, Nandor; Dorronsoro, Carlos

    2014-02-01

    The combination of air-puff systems with real-time corneal imaging (i.e. Optical Coherence Tomography (OCT), or Scheimpflug) is a promising approach to assess the dynamic biomechanical properties of the corneal tissue in vivo. In this study we present an experimental system which, together with finite element modeling, allows measurements of corneal biomechanical properties from corneal deformation imaging, both ex vivo and in vivo. A spectral OCT instrument combined with an air puff from a non-contact tonometer in a non-collinear configuration was used to image the corneal deformation over full corneal cross-sections, as well as to obtain high speed measurements of the temporal deformation of the corneal apex. Quantitative analysis allows direct extraction of several deformation parameters, such as apex indentation across time, maximal indentation depth, temporal symmetry and peak distance at maximal deformation. The potential of the technique is demonstrated and compared to air-puff imaging with Scheimpflug. Measurements ex vivo were performed on 14 freshly enucleated porcine eyes and five human donor eyes. Meas