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PubMed2000

Changes in epidermal growth factor receptors in olfactory epithelium associated with aging.

To clarify the mechanisms of age-related changes in the olfactory epithelium, we investigated age-related changes in epidermal growth factor receptors (EGFRs) in the epithelium. We examined 3 mice at each of the following stages: embryonal (embryonic days 14 and 16), neonatal (postnatal days 1 and 7), adult (postnatal weeks 5 and 12), and aged (postnatal years 1.5 to 2). The olfactory epithelium of each mouse was stained with sheep anti-human EGFR polyclonal IgG by an immunohistochemical method. The EGFRs were observed in all layers of the olfactory epithelium at the embryonal and neonatal stages. They were identified only in the basal layer of the olfactory epithelium at adult and aged stages, and the number of regions in which EGFRs were identified in the basal layer of the olfactory epithelium decreased in aged mice compared to adult mice. We believe that a decrease in EGFRs in the olfactory epithelium induces the inhibition of cell proliferation, with resultant atrophy of the olfactory epithelium.

Aging↗

PubMed2006

Tissue engineering for regeneration of the tracheal epithelium.

OBJECTIVES: The slowness of epithelialization on the artificial trachea that has been successfully used in humans is a problem. The purpose of this study was to develop a way to regenerate the epithelium on the surface of this artificial trachea. METHODS: In an in vitro study, isolated rat tracheal epithelial cells were seeded on a collagenous gel that was stratified on a collagenous sponge. Histologic and immunohistochemical examinations were made. In an in vivo study, we transplanted grafts with green fluorescent protein-positive tracheal epithelial cells onto the tracheal defects of normal rats. At 3, 7, 14, and 30 days after the operation, histologic and immunohistochemical examinations were made. RESULTS: In the in vitro study, the 3 layers--the epithelium, gel, and sponge--could be observed. The epithelium expressed cytokeratin 14, cytokeratin 18, and occludin. In the in vivo study, the artificial trachea was covered with epithelium at 3 days after operation, and then the epithelium differentiated from single- or double-stratified squamous epithelium into columnar ciliated epithelium. Green fluorescent protein-positive cells were found 3 days after operation. CONCLUSIONS: We believe that the method used in our experiment is an effective way to regenerate the epithelium on the surface of an artificial trachea. With further experimentation, this method should be suitable for clinical application.

Animals↗

PubMed2000

The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium.

The embryonic gut of vertebrates consists of endodermal epithelium, surrounding mesenchyme derived from splanchnic mesoderm and enteric neuronal components derived from neural crest cells. During gut organogenesis, the mesenchyme differentiates into distinct concentric layers around the endodermal epithelium forming the lamina propria, muscularis mucosae, submucosa and lamina muscularis (the smooth muscle layer). The smooth muscle layer and enteric plexus are formed at the outermost part of the gut, always some distance away from the epithelium. How this topographical organization of gut mesenchyme is established is largely unknown. Here we show the following: (1) Endodermal epithelium inhibits differentiation of smooth muscle and enteric neurons in adjacent mesenchyme. (2) Endodermal epithelium activates expression of patched and BMP4 in adjacent non-smooth muscle mesenchyme, which later differentiates into the lamina propria and submucosa. (3) Sonic hedgehog (Shh) is expressed in endodermal epithelium and disruption of Shh-signaling by cyclopamine induces differentiation of smooth muscle and a large number of neurons even in the area adjacent to epithelium. (4) Shh can mimic the effect of endodermal epithelium on the concentric stratification of the gut. Taken together, these data suggest that endoderm-derived Shh is responsible for the patterning across the radial axis of the gut through induction of inner components and inhibition of outer components, such as smooth muscle and enteric neurons.

Animals↗

PubMed2003

Expression of retinoblastoma gene product in respiratory epithelium and sinonasal neoplasms: relationship with p16 and cyclin D1 expression.

Transition from G1 to S phase of the cell cycle is mediated by interactions between the Retinoblastoma gene product (pRb), p16, and cyclin D1. To determine the expression of these proteins in the sinonasal mucosa immunohistochemistry was carried out on archived tissue sections from 46 patients (37 men, 9 women, age range 17 to 82 years, median 55 years). Nuclear immunostaining for these proteins was assessed and the expression rates (percentages of immunoreactive nuclei) in normal respiratory epithelium, inverted sinonasal papillomas, cylindrical (oncocytic) sinonasal papillomas, and squamous cell carcinomas were compared. Normal respiratory epithelium showed significantly higher pRb expression in surface cells compared to basal cells (p < 0.05). In contrast, abundant pRb expression in surface and basal cells was detected in columnar differentiation in sinonasal papillomas and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed significantly reduced pRb expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05, respectively). Expression of p16 was detected in all epithelial cell layers of normal respiratory epithelium, sinonasal papillomas, and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed increased p16 expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05 between squamous metaplasia and columnar epithelium). Sinonasal squamous cell carcinomas showed the coexpression of pRb and p16. Expression rates of cyclin D1 higher than 10% were detected only in invasive carcinomas but not in carcinoma in situ, sinonasal papillomas or respiratory epithelium. Conclusively, pRb expression accompanies terminal differentiation in columnar surface cells. Expression of pRb in proliferating basal cells is present in sinonasal papillomas and adjacent mucosa but not in normal respiratory epithelium. Cuboidal and squamous metaplasia in inverted papillomas involves downregulation of pRb expression along with increased p16 expression in surface cells. Sinonasal squamous cell carcinomas coexpress pRb and p16. Overexpression of cyclin D1 in sinonasal lesions is confined to invasive squamous cell carcinomas.

Carcinoma, Squamous Cell↗

PubMed1999

Multi-layered periodontal pocket epithelium reconstituted in vitro: histology and cytokeratin profiles.

BACKGROUND: In order to study inter-individual differences in bacterial adhesion/invasion of periodontal tissues, an in vitro model for culturing multi-layered pocket epithelium without feeder layers or stromal equivalents (including the evaluation of their cytokeratin profiles) was developed. METHODS: Pocket epithelium was collected and grown until confluent in Falcon flasks using keratinocyte-serum free medium (KSFM), without a feeder layer. In the second passage, oral keratinocytes were re-grown in a 2 compartment system using either a clear polyester (transwell-clear [TCL]) or a collagen (transwell-col [TCO]) membrane as culture surface. After the first week, the calcium concentration was raised to 1.2 mM and in half the wells, the KSFM was supplemented with 10% fetal calf serum (FCS). Histology and immunohistochemistry were performed after 1, 2, and 3 weeks of additional growth. RESULTS: In general, all conditions resulted in a structured epithelium consisting of 3 to 5 layers, but important differences were observed between the membrane types and between the media. CK4 was rarely and only lightly expressed while CK18 and 19 (characteristic of junctional epithelium) were very strongly expressed in the older (2 and 3 weeks) cultures. CK13 and 14 (characteristic of any stratifiable epithelial cell) also tended to increase over time; CK13 seemed to be stronger in KSFM with FCS while the contrary was true for CK14. The multi-layer created by the combination TCL/KSFM + 10% FCS resembled a junctional epithelium most, while that grown on TCO without FCS mimicked the sulcular epithelium. CONCLUSIONS: It seems possible to create a histiotypic culture resembling either periodontal pocket or junctional epithelium without the use of stromal equivalents or feeder layers which make this approach more cumbersome. This multi-layered culture offers a model to investigate the permeability of pocket epithelium and the adhesion and penetration of bacteria under well-defined environmental conditions.

Animals↗

PubMed2000

Airway epithelium wound repair and regeneration after injury.

The airway epithelium represents the first line of lung defense through different mechanisms such as mucociliary clearance, ion secretion, water regulation and secretion of antibacterial, antiprotease, antioxidant and anti-inflammatory molecules. The intercellular junctional complexes also participate to the preservation of the airway epithelium integrity. Nevertheless, because of its permanent contact with the external milieu, the airway epithelium is frequently injured. Immediately after injury, the airway epithelium initiates a wound healing process to restore its barrier integrity. Several in vitro, ex vivo and in vivo assays have been described to analyze the different steps of wound repair following airway cell damaging. The epithelial process of repair following a wound created in vitro by mechanical or chemical injury is similar to that observed in vivo in animal models. The first and most important event occurring after denudation of the airway epithelium is cell migration and not proliferation. The airway epithelial cells migrate without any external stimulation or non-epithelial cell participation at a speed of about 30-40 microns/h. The cellular and molecular factors involved in wound repair and epithelial regeneration are closely interacting and imply a coordinated sequence of events involving: alteration of the actin cytoskeleton, secretion of extracellular matrix proteins and expression of integrin receptors. Recent human airway xenografts have been developed which have demonstrated that adult airway cells are capable of regenerating a well-differentiated and functional epithelium after successive steps that mimic regeneration after airway epithelium injury. The matrix metalloproteinases are determinant factors in tissue repair by remodeling extracellular matrix and by releasing growth factors. The specific involvement of growth factors such as hepatocyte growth factor in enhancing airway regeneration and repair suggests that these molecules may find future pharmacological application in the treatment of injured airway epithelium.

Cell Communication↗

PubMed2001

Histopathology of the airway epithelium in an experimental dual-phase model of bronchial asthma.

BACKGROUND: Lesions of trachea cuticles are a pathological histological characteristic of bronchial asthma. Furthermore, collected tracheal cuticles desquamated from the respiratory tract are found in patients' sputum when asthma attacks occur or after the induction of allergen inhalation. From these facts, it is assumed that desquamation of trachea cuticle cells is a pathological symptom of bronchial asthma. However, there has not been any chronological report of desquamation of trachea cuticles through the process of bronchial asthma attacks. OBJECTIVE: For this report, we made an experimental bronchial asthma model using guinea pigs, and conducted chronological examinations of trachea cuticle lesions related to pathological symptoms of bronchial asthma using a transmission electron microscope and a scanning electron microscope. METHODS: The experimental asthma models were made by injection of ovalbumin into the abdominal cavity of guinea pigs. Then the airway responses to inhaled aerosolized ovalbumin were induced. The trachea were enucleated and examined under an optical microscope, a transmission electron microscope (hereafter abbreviated as TEM), and a scanning electron microscope (hereafter abbreviated as SEM) after 1, 2, 4, 8, 12, 24 h and 7 days after the immediate airway responses. RESULTS: Intercellular oedema of ciliated epithelium was observed in the sensitization groups immediately after the immediate airway response. SEM observation revealed increased mucus secretion and shortening of cilium. A slight case of desquamation or deciduation of ciliated epithelium was also beginning to appear. TEM observation revealed a dilation of ciliated epithelium intervals. Infiltration of eosinophilic leucocytes was already detectable beneath the ciliated epithelium. The degree of ciliated epithelium desquamation and infiltration of eosinophilic leucocytes progressed with time. When the late airway response occurred 4 hours later, eosinophilic leucocytes had increased drastically, and ciliate epithelium had desquamated to the extent that basal cells were exposed. Seven days after the immediate airway response, epithelium intercellular oedema had improved, and cilium had been reproduced. CONCLUSION: These results suggest that desquamation of epithelium caused by trachea cuticle lesions appears at an early stage of an asthma attack, owing to the contraction of the trachea, and that the damage is intensified by the infiltration of eosinophilic leucocytes.

Animals↗

PubMed1975

[Morphological changes in the follicular epithelium in oogenesis of the Peking duck (Anas boschas domestica) and other poultry].

The morphology of the follicular epithelium during the course of oogenesis in poultry (duck goose, hen, turkey) and at the first stages of oocyte growth in some wild birds (finch, totmit, wood-pecker, pigeon) was studied. The general patterns of the follicular epithelium changings are similar in the both groups of birds. In the process of the oocyte growth the flat follicular epithelial monolayer changes to cubic, prismatic, pseudostratified epithelium. It leads sometimes to the impression of multi-layerness owing to its irregular structure. During the oocyte rapid growth the surface of the oocyte increases causing tension on the epithelium layer. This process again turns the pseudostratified epithelium into primatic, cubic and flat epithelium. Pseudostratified structure of the follilicular epithelium is regarded as adaptation to the necessity of the rapid tension during rapid oocyte growth. Correlations of the follicular epithelium morphology with the oocyte diameter are established. Existance of a temporary multilayer stage is discussed with arguments against the interpretation of this stage as a real multilayer.

Animals↗

PubMed2002

Aberrant promoter methylation in bronchial epithelium and sputum from current and former smokers.

Recent studies from our laboratory suggest that gene-specific methylation changes in sputum could be good intermediate markers for the early detection of lung cancer and defining the efficacy of chemopreventive interventions. The purpose of our study was to determine the prevalence for aberrant promoter methylation of the p16, O(6)-methylguanine-DNA methyltransferase (MGMT), death-associated protein (DAP) kinase, and Ras effector homologue (RASSFIA) genes in nonmalignant bronchial epithelial cells from current and former smokers in a hospital-based, case control study of lung cancer. The relationship between loss of heterozygosity, at 9p and p16 methylation in bronchial epithelium and the prevalence for methylation of these four genes in sputum from cancer-free, current and former smokers were also determined. Aberrant promoter methylation of p16 was seen in at least one bronchial epithelial site from 44% of cases and controls. Methylation of the DAP kinase gene was seen in only 1 site from 5 cases and 4 controls, whereas methylation of the RASSFIA was not detected in the bronchial epithelium. Promoter methylation for p16 and DAP kinase was seen as frequently in bronchial epithelium from current smokers as from former smokers. No promoter methylation of these genes was detected in bronchial epithelium from never-smokers. Methylation of the p16 gene was detected in sputum from 23 of 66 controls. DAP kinase gene promoter methylation was also seen in sputum from 16 controls, and 8 of these subjects were positive for p16 methylation. Methylation of the MGMT gene was seen in sputum from 9 controls, whereas RASSFIA promoter methylation was only seen in 2 controls. The correlation between p16 status in the bronchial epithelium obtained from lung lobes that did not contain the primary tumor and the tumor itself was examined. Seventeen of 18 tumors (94%) showed an absolute concordance, being either methylated in the tumor and at least 1 bronchial epithelial site, or unmethylated in both tumor and bronchial epithelium. These results indicate that aberrant promoter hypermethylation of the p16 gene, and to a lesser extent, DAP kinase, occurs frequently in the bronchial epithelium of lung cancer cases and cancer-free controls and persists after smoking cessation. The strong association seen between p16 methylation in the bronchial epithelium and corresponding primary tumor substantiates that inactivation of this gene, although not transforming by itself, is likely permissive for the acquisition of additional genetic and epigenetic changes leading to lung cancer.

Adult↗

PubMedDate not supplied

3p21, 5q21, 9p21 and 17p13.1 allelic deletions are potential markers of individuals with a high risk of developing adenocarcinoma in Barrett's epithelium without dysplasia.

BACKGROUND/AIMS: A common genetic abnormality detected in Barrett's adenocarcinoma is LOH (loss of heterozygosity) at the sites of known or putative tumor suppressor genes. Thus, some deletions have also been determined in peritumoral Barrett's epithelium. These findings suggest that a tissue field of somatic genetic alterations precede the histopathological phenotypic changes of carcinoma. We investigated 32 cases of Barrett's esophagus with no evidence of dysplasia for LOH at 5q21 (APC), 3p21, 9p21 (p16) and 17p13.1 (p53) chromosomal regions. METHODOLOGY: Two groups were randomly selected and compared: 16 cases of Barrett's epithelium adjacent to adenocarcinoma and 16 cases of Barrett's epithelium with no evidence of malignant transformation in a 5-10 years follow-up period. In three adenocarcinomas cases several previous endoscopic biopsies of Barrett's esophagus were available. RESULTS: We determined frequent allelic losses in adenocarcinomas at p53 (54%), p16 (50%), 3p21 (40%) and 5q21 (33%). Identical LOH was present in most cases in the Barrett's epithelium adjacent to adenocarcinoma. LOH at these loci was unusual in Barrett's epithelium with no evidence of malignant transformation. However, in cases where sequential endoscopic biopsies were performed in advance to the adenocarcinoma diagnosis LOH was already present in the Barrett's epithelium. CONCLUSIONS: We suggest that LOH at these loci may be present before the onset of the malignant growth and LOH studies may supplement the histopathological evaluation of Barrett's epithelium. LOH at 3p21, 5q21, 9p21 and 17p13 chromosomal regions in cells of Barrett's epithelium without dysplasia may have a role as a potential marker for individuals with a high risk of developing adenocarcinoma.

Adenocarcinoma↗

PubMed2006

Some markers of proliferative activity in cholesteatoma epithelium in adults.

BACKGROUND: The aim of this study was to determine the proliferative capacity in certain layers of the cholesteatoma epithelium and normal skin. MATERIAL/METHODS: Cholesteatoma and skin were obtained during ear surgical procedures. The number of PCNA- and Ki-67-positive cells were counted within each of three microscopic fields at a magnification of x400. Fourteen normal skin specimens collected from the retroauricular area were used as controls. RESULTS: The skin specimens were found to have a lower proliferative capacity than the cholesteatoma epithelium. This activity was mainly observed in the cell nuclei of the basal layer of the epidermis. The percentage of PCNA-positive cells was 23% in the epidermis and 45.7% in the cholesteatoma epithelium. Immunohistochemical investigation of the epidermis revealed 7% Ki-67-positive cells, mainly located in the basal layer of the epidermis. The presence of Ki-67 antigen in the cell nuclei was determined in the parabasal layer of the cholesteatoma epithelium and stroma. Ki-67-positive cells made up 22% of the cholesteatoma epithelium, compared with 7% in the skin. CONCLUSIONS: Proliferative activity is mainly observed in the basal and suprabasal layers of the cholesteatoma epithelium. The proliferative capacity of the cholesteatoma epithelium is different in the various areas of the epithelium and does not depend on its thickness.

Adult↗

PubMed1991

[Mitotic activity of rabbit corneal epithelium in vitro].

It is difficult to maintain corneal epithelial cells for several passages in culture. At a first step to establish a culture system for corneal epithelium, the authors evaluated factors which seemed to be involved in the growth kinetics and mitotic activity of rabbit corneal epithelium in vitro. First of all, to compare the growth kinetics of peripheral and central epithelium, we measured the areas and distances of the epithelium that outgrew from peripheral and central explants in culture. The peripheral corneal epithelium grew 1.2-to 1.6-fold better than the central corneal epithelium. However, there was no significant difference between them. Secondly, we evaluated the effects of epidermal growth factor (EGF), cholera toxin (CTX) and insulin, which were contained in supplemented hormonal epithelial medium (SHEM) developed by Jumblatt et al, on the mitotic activity of rabbit corneal epithelium. Among these drugs, only EGF had the ability to increase the epithelial cell number after 10 days of incubation. By contrast, CTX which elevated the level of cyclic AMP inhibited the proliferation of the epithelium.

Animals↗

PubMed1996

Fabrication of cultured epithelium using oral mucosal cells and its clinical applications.

A cultured mucosal epithelium can be formed with living mucosal cells in vitro and used as a graft material. In this article, we described our culture methods for preparation of cultured mucosal epithelium as well as its biological characteristics compared with cultured skin epithelium. According to our results, cultured mucosal epithelium was formed earlier than cultured skin epithelium. Furthermore, the structure of cultured mucosal epithelium was maintained longer than that of cultured skin epithelium. Based on our findings from an in vitro study, we applied this cultured mucosal epithelium to the reconstruction of human skin and mucosal defects and succeeded in repairing the defects. We also present an overview of the problem relevant to the use of such methods.

3T3 Cells↗

PubMed1997

Immunolocalization of proliferating cell nuclear antigen in the peri-implant epithelium.

The aim of the present study was to investigate the proliferating activity of peri-implant epithelium immunohistochemically using proliferating cell nuclear antigen (PCNA). Eight ITI (Internationale Team für Implantologie) implants were placed into simulated sockets in the mandibles of two beagle dogs two months following tooth extraction. As a control, junctional epithelium of the molar teeth in the same animals were used. The nature of staining and the distribution of PCNA immunoreactivity were determined by scoring a minimum of 100 cells on two sections from each of the implants. In the junctional epithelium, the immunoreactivity to PCNA was detected mainly in the basal cells, in some of the prickle cells, and in a few cells attached to the enamel. In peri-implant epithelium, only some of the basal cells were positive for PCNA. The PCNA score of the peri-implant epithelium was significantly lower than that of junctional epithelium. These results suggest that the peri-implant epithelium maintains a lower capacity to act as a proliferative defence mechanism than does the junctional epithelium.

Animals↗

PubMed1990

Whole population cell kinetics of jejunal and colonic epithelium in lactating dams.

Previous studies make it likely that the response of the intestinal epithelium as a whole to lactation is different from that observed in the crypt population alone. We confirm this difference by whole population cell kinetics measurements of jejunal and colonic epithelium in mice that have been suckling pups for various lengths of time. We found that the fraction of cells in S phase in jejunal epithelium was significantly increased after only 1 week of lactation, maintained this elevated level after 2 weeks of lactation, but returned to normal during the third week of lactation. The cell number density in jejunum was also significantly higher after 2 and 3 weeks of lactation before returning to normal by 4 weeks. In the colonic epithelium no changes were found in the distribution of cells in G1, S, and G2 + M phases. However, a significant increase in cell number density was observed after 2 weeks of lactation, followed by a sharp decrease to a level significantly below that of normal mice after 3 and 4 weeks of lactation. We conclude that the observed significant increase in the fraction of S phase in jejunal epithelium of lactating mice is probably due to a smaller relative expansion of the villus population when compared with the expansion of the crypt population. Our data also indicate that a number of cell kinetic parameters in the intestinal epithelium of lactating mice are changing throughout the period of lactation. Thus the intestinal epithelium is probably not in a steady state during lactation.

Animals↗

PubMed1995

Ectopic TGF beta 1 expression in the secretory mammary epithelium induces early senescence of the epithelial stem cell population.

An important feature of the mammary gland is the regenerative capacity of its epithelium which is demonstrated upon successive cycles of lactation and involution. Pregnant mice expressing a whey-acidic protein (WAP) promoter-driven transforming growth factor-beta 1 (TGF beta 1) cDNA are unable either to generate a secretory mammary epithelium or to lactate. Here we investigate whether ectopic TGF beta 1 induces this phenotype by affecting the transgenic epithelium directly or in trans. Reciprocal transplantation of mammary tissue between normal and transgenic hosts resulted in the development of the respective phenotypes of the transplants within the same mammary fat pad. When isolated mammary epithelial cells from both were mixed before implantation so that transgenic and normal epithelium would develop together more proximately, both phenotypes were simultaneously observed in the resultant chimeric mammary outgrowths. Since no trans effect was detectable, we hypothesize that early expression of the transgene results in compromised lobular progenitor cells through an intracrine mechanism. Consistent with this posit, WAP promoter-driven protein expression was detected in individual cells of the subtending ducts of immature females at estrus. Transplantation of WAP-TGF beta 1 mammary gland into nonpregnant hosts revealed that transgenic implants, even those from young postpubertal virgin females, had a diminished ability to repopulate epithelium-free mammary fat pads. Accordingly, the ectopic expression of WAP-TGF beta 1 not only impairs lobular progenitors, but also promotes an early senescence of the regenerative capacity of the mammary ductal epithelium. This leads us to propose that mammary epithelial stem cells give rise to two functionally distinct progenitor cells in the mammary gland epithelium: one capable of producing daughters committed to ductal formation, the other capable only of producing daughters committed to lobular function.

Animals↗

PubMed1996

Transport of circulating reduced glutathione at the basolateral side of the anterior lens epithelium: physiologic importance and manipulations.

Transport of circulating reduced glutathione (GSH) was studied at the basolateral side of the lens epithelium by using an in situ vascular eye perfusion technique in guinea-pigs with rapid sampling to ensure detecting initial uptakes. The unidirectional transport rates of [35S]-GSH (4 nM) from plasma and aqueous into the epithelial cytosol were 0.046 +/- 0.003 and 6.88 +/- 0.39 min-1, respectively. HPLC analysis indicated that over 94% of [35S]-GSH remained intact in the epithelium and cortex in the presence or absence of gamma-glutamyl transpeptidase inhibitor, serine borate. Simultaneous infusion of [35S-cysteine]-GSH and [3H-glycine]-GSH confirmed the non-involvement of gamma-glutamyl transpeptidase in GSH transport across the lenticular membranes by showing that 35S/3H ratio in the epithelium and cortex was the same as in the aqueous and plasma. GSH epithelial influx was reduced by 53% (P < 0.01) by 0.3 mM sulphobromophtalein-GSH, a GSH conjugate that does not inhibit the facilitative GSH transporter, RcGshT, recently found in the lens. At physiologic concentration of circulating GSH at 30 microM, GSH epithelial influx was 0.77 nmol min-1 g-1; a t1/2 of 85.4 hr was estimated if endogenous epithelial GSH had to be replaced exclusively by plasma-derived GSH. The level of GSH in the epithelium was increased by 38% (P < 0.05) by 1 hr arterial infusion of GSH at 20 mM. The aqueous concentration of GSH under these conditions was 1.2 mM so that accumulation in the epithelium occurred a greater than six-fold concentration gradient. It is concluded that: (a) transport of GSH at the basolateral side of the epithelium is mediated by a concentrative mechanism distinct from RcGshT: (b) circulating GSH may represent a major source for epithelial GSH under physiologic conditions; and (c) the level of GSH in the epithelium can be manipulated by exogenous GSH.

Animals↗

PubMed1997

Tissue culture of retinal pigment epithelium following isolation with a gelatin matrix technique.

Prior to transplantation of the retinal pigment epithelium, it is necessary to develop techniques to harvest viable retinal pigment epithelium as an organized monolayer. Unfortunately, current techniques result in contraction of the harvested monolayer and coiling of the cell sheets, which hinders successful transplantation. The purpose of this study was to develop a method for harvesting retinal pigment epithelium from eye cups and from tissue culture prior to transplantation. Passage 1 porcine retinal pigment epithelium and native retinal pigment epithelium from fresh porcine eye cups were pretreated with 0.25% edetic acid for 12 minutes and coated with a 100 micron layer of 12% gelatin. Patches of the retinal pigment epithelial cell monolayer were harvested and transferred to culture plates, and cell viability and the ability of the transferred cells to proliferate in culture was determined. Our results demonstrated that retinal pigment epithelium can be harvested from tissue culture and eye cups as an organized monolayer with high efficiency (94.7 +/- 3.5% and 99.7 +/- 0.3% harvesting rates, respectively) and high cell viability (91.3 +/- 2.9% and 89.4 +/- 4.3%, respectively). Cells harvested from tissue culture plates divided and became confluent within 10 to 14 days. Cells harvested from eye cups maintained a differentiated phenotype and migrated outward from the margin of the exoplant. There was no contraction of the retinal pigment epithelial monolayer isolated from either substrate. Thus, we were able to harvest retinal pigment epithelium as an organized monolayer from tissue culture plates and freshly enucleated eyes with edetic acid and gelatin. The harvested cells were viable and proliferated without contraction of the monolayer in vitro.

Animals↗