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Charles R Wira - One of the best experts on this subject based on the ideXlab platform.
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2003. Effect of mouse uterine stromal cells on epithelial cell Transepithelial Resistance (TER) and TNFalpha and TGFbeta release in culture. Biol. Reprod
2016Co-Authors: Katherine S Grant, Charles R WiraAbstract:Recognizing that uterine stromal cells regulate several uter-ine epithelial cell function(s), the current study was undertak-en to more fully define cell-cell communication in the uterus and to examine the role of uterine stromal cells in regulating epithelial cell monolayer integrity and cytokine release. Uter-ine epithelial and stromal cells from adult intact mice were isolated and cultured separately on cell culture inserts and/or in culture plates. Epithelial cells, which reach confluence as indicated by high Transepithelial Resistance (TER. 1000 ohms/ well), preferentially release transforming growth factor-beta (TGFb) into the basolateral chamber (ø70 %. apical) and tu-mor necrosis factor-alpha (TNFa) into the apical compartment (ø30 %. basolateral). When epithelial cells on cell culture inserts were transferred to plates containing stromal cells, co-culture for 24–48 h increased epithelial cell TER (ø70 % higher than control) and decreased TNFa release into both the apical and basolateral chambers (ø30%–50%). In contrast, TGFb re-lease was not affected by the presence of stromal cells. In other studies, the effects of stromal cells on epithelial cell TER and TNFa release persisted for 5–7 days following the removal of stromal cells and were also seen in coculture studies in which conditioned stromal media (CSM) was placed in the basolateral chamber. These studies indicate that uterine stromal cells pro-duce a soluble factor(s) that regulates epithelial cell TER and release of TNFa without effecting TGFb release. These results suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to maintain uterine integrity and epithelial secretory function. cytokines, female reproductive tract, immunology, uteru
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paracrine mediators of mouse uterine epithelial cell Transepithelial Resistance in culture
Journal of Reproductive Immunology, 2005Co-Authors: Katherine S Granttschudy, Charles R WiraAbstract:Epithelial cell integrity at mucosal surfaces provides an effective physical barrier against potential pathogens that threaten reproductive health. We have used polarized epithelial cells from adult mouse uteri to investigate the roles of TNFalpha and TGFbeta, which are produced by uterine epithelial and stromal cells, and hepatocyte growth factor (HGF), produced by uterine stromal cells, in regulating epithelial cell integrity measured as Transepithelial electrical Resistance (TER). Exposure of epithelial cells to TNFalpha, TGFbeta, and HGF have profound effects on TER that are different from their known actions on TER at other mucosal surfaces. When incubated with TNFalpha, TER increased in a dose-dependent manner. In contrast, when cells were incubated with TGFbeta, TER was markedly but reversibly suppressed. Interestingly, HGF, when placed in the basolateral compartment, increased TER. Based on these findings, we conclude that TNFalpha, TGFbeta and HGF may play regulatory roles in modulating epithelial cell tight junctions. These studies suggest that factors, such as hormone balance, pathogen exposure as well as pregnancy, which affect cytokine and growth factor secretion, influence epithelial cell barrier protection within the female reproductive tract.
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hepatocyte growth factor regulation of uterine epithelial cell Transepithelial Resistance and tumor necrosis factor α release in culture
Biology of Reproduction, 2005Co-Authors: Katherine S Granttschudy, Charles R WiraAbstract:Abstract Underlying stromal cells are essential for the normal development of epithelial cells (ECs) at mucosal surfaces. Recent studies from our laboratory have shown that uterine stromal cells regulate EC integrity, measured as Transepithelial Resistance (TER) as well as tumor necrosis factor (TNF) α α secretion by ECs in culture. Using stromal cells in coculture with polarized ECs grown on inserts, we found that stromal cells produce soluble mediators that increase TER and decrease TNFα secretion. The purpose of the present study was to identify the mechanisms whereby stromal cells exert their effects on uterine epithelium. We report that hepatocyte growth factor (HGF), a known mesenchymal growth factor that mediates EC proliferation, increases TER but, at the same time, decreases apical TNFα release. When ECs and/or stromal cells were incubated with anti-HGF or anti-HGF receptor (HGFR) antibody before HGF, the effects of HGF were blocked. These findings indicate that ECs express the HGFR at their baso...
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effect of estradiol on mouse uterine epithelial cell Transepithelial Resistance ter
American Journal of Reproductive Immunology, 2004Co-Authors: Katherine S Granttschudy, Charles R WiraAbstract:Problem: The effects of estradiol on epithelial cell function in the uterus may either be direct or indirect through the paracrine effects of underlying stromal cells. The aim of this study was to test whether estradiol-17β (E2) acts directly to regulate uterine epithelial cell monolayer integrity. Methods of Study: Mouse uterine epithelial cells were isolated and grown on cell culture inserts to form confluent, polarized monolayers, as indicated by the development of high Transepithelial Resistance (TER). Results: When polarized epithelial cells were treated with E2, TER was significantly decreased within 24 hr of exposure. Epithelial cells remained hormonally responsive in culture for at least 10 days. In contrast to estradiol, incubation with progesterone, cortisol, aldosterone, and DHT had no effect on uterine epithelial cell TER. The ability of E2 to decrease TER was inhibited following co-incubation with ICI 182,780, a pure estrogen receptor antagonist. To further investigate the mechanism involved in estradiol-induced decreases in TER, we tested the effect of TAPI-0, an inhibitor of matrix metalloproteinases. Our findings indicate that TAPI-0 reversed the inhibitory effect of E2 on TER. Conclusions: These studies demonstrate that epithelial monolayer integrity is directly influenced by E2 and ER mediated. Further, it suggests that the mechanism through which estradiol decreases TER is mediated by matrix metalloproteinases.
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effect of mouse uterine stromal cells on epithelial cell Transepithelial Resistance ter and tnfα and tgfβ release in culture
Biology of Reproduction, 2003Co-Authors: Katherine S Grant, Charles R WiraAbstract:Abstract Recognizing that uterine stromal cells regulate several uterine epithelial cell function(s), the current study was undertaken to more fully define cell-cell communication in the uterus and to examine the role of uterine stromal cells in regulating epithelial cell monolayer integrity and cytokine release. Uterine epithelial and stromal cells from adult intact mice were isolated and cultured separately on cell culture inserts and/or in culture plates. Epithelial cells, which reach confluence as indicated by high Transepithelial Resistance (TER > 1000 ohms/well), preferentially release transforming growth factor-beta (TGFβ) into the basolateral chamber (≈70% > apical) and tumor necrosis factor-alpha (TNFα) into the apical compartment (≈30% > basolateral). When epithelial cells on cell culture inserts were transferred to plates containing stromal cells, coculture for 24–48 h increased epithelial cell TER (≈70% higher than control) and decreased TNFα release into both the apical and basolateral chambe...
Katherine S Granttschudy - One of the best experts on this subject based on the ideXlab platform.
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paracrine mediators of mouse uterine epithelial cell Transepithelial Resistance in culture
Journal of Reproductive Immunology, 2005Co-Authors: Katherine S Granttschudy, Charles R WiraAbstract:Epithelial cell integrity at mucosal surfaces provides an effective physical barrier against potential pathogens that threaten reproductive health. We have used polarized epithelial cells from adult mouse uteri to investigate the roles of TNFalpha and TGFbeta, which are produced by uterine epithelial and stromal cells, and hepatocyte growth factor (HGF), produced by uterine stromal cells, in regulating epithelial cell integrity measured as Transepithelial electrical Resistance (TER). Exposure of epithelial cells to TNFalpha, TGFbeta, and HGF have profound effects on TER that are different from their known actions on TER at other mucosal surfaces. When incubated with TNFalpha, TER increased in a dose-dependent manner. In contrast, when cells were incubated with TGFbeta, TER was markedly but reversibly suppressed. Interestingly, HGF, when placed in the basolateral compartment, increased TER. Based on these findings, we conclude that TNFalpha, TGFbeta and HGF may play regulatory roles in modulating epithelial cell tight junctions. These studies suggest that factors, such as hormone balance, pathogen exposure as well as pregnancy, which affect cytokine and growth factor secretion, influence epithelial cell barrier protection within the female reproductive tract.
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hepatocyte growth factor regulation of uterine epithelial cell Transepithelial Resistance and tumor necrosis factor α release in culture
Biology of Reproduction, 2005Co-Authors: Katherine S Granttschudy, Charles R WiraAbstract:Abstract Underlying stromal cells are essential for the normal development of epithelial cells (ECs) at mucosal surfaces. Recent studies from our laboratory have shown that uterine stromal cells regulate EC integrity, measured as Transepithelial Resistance (TER) as well as tumor necrosis factor (TNF) α α secretion by ECs in culture. Using stromal cells in coculture with polarized ECs grown on inserts, we found that stromal cells produce soluble mediators that increase TER and decrease TNFα secretion. The purpose of the present study was to identify the mechanisms whereby stromal cells exert their effects on uterine epithelium. We report that hepatocyte growth factor (HGF), a known mesenchymal growth factor that mediates EC proliferation, increases TER but, at the same time, decreases apical TNFα release. When ECs and/or stromal cells were incubated with anti-HGF or anti-HGF receptor (HGFR) antibody before HGF, the effects of HGF were blocked. These findings indicate that ECs express the HGFR at their baso...
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effect of estradiol on mouse uterine epithelial cell Transepithelial Resistance ter
American Journal of Reproductive Immunology, 2004Co-Authors: Katherine S Granttschudy, Charles R WiraAbstract:Problem: The effects of estradiol on epithelial cell function in the uterus may either be direct or indirect through the paracrine effects of underlying stromal cells. The aim of this study was to test whether estradiol-17β (E2) acts directly to regulate uterine epithelial cell monolayer integrity. Methods of Study: Mouse uterine epithelial cells were isolated and grown on cell culture inserts to form confluent, polarized monolayers, as indicated by the development of high Transepithelial Resistance (TER). Results: When polarized epithelial cells were treated with E2, TER was significantly decreased within 24 hr of exposure. Epithelial cells remained hormonally responsive in culture for at least 10 days. In contrast to estradiol, incubation with progesterone, cortisol, aldosterone, and DHT had no effect on uterine epithelial cell TER. The ability of E2 to decrease TER was inhibited following co-incubation with ICI 182,780, a pure estrogen receptor antagonist. To further investigate the mechanism involved in estradiol-induced decreases in TER, we tested the effect of TAPI-0, an inhibitor of matrix metalloproteinases. Our findings indicate that TAPI-0 reversed the inhibitory effect of E2 on TER. Conclusions: These studies demonstrate that epithelial monolayer integrity is directly influenced by E2 and ER mediated. Further, it suggests that the mechanism through which estradiol decreases TER is mediated by matrix metalloproteinases.
José-alain Sahel - One of the best experts on this subject based on the ideXlab platform.
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Chronic exposure to tumor necrosis factor alpha induces retinal pigment epithelium cell dedifferentiation
Journal of Neuroinflammation, 2018Co-Authors: Sara Touhami, Thibaud Mathis, Fanny Beguier, Sacha Reichman, Sébastien Augustin, Hugo Charles-messance, Lucile Vignaud, Emeline F. Nandrot, Valérie Forster, José-alain SahelAbstract:Background The retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important barrier and immuno-suppressive functions in the eye. We have previously shown that acute stimulation of RPE cells by tumor necrosis factor alpha (TNFα) downregulates the expression of OTX2 (Orthodenticle homeobox 2) and dependent RPE genes. We here investigated the long-term effects of TNFα on RPE cell morphology and key functions in vitro. Methods Primary porcine RPE cells were exposed to TNFα (at 0.8, 4, or 20 ng/ml per day) for 10 days. RPE cell morphology, phagocytosis, barrier- and immunosuppressive-functions were assessed. Results Chronic (10 days) exposure of primary RPE cells to TNFα increases RPE cell size and polynucleation, decreases visual cycle gene expression, impedes RPE tight-junction organization and Transepithelial Resistance, and decreases the immunosuppressive capacities of the RPE. TNFα-induced morphological- and Transepithelial-Resistance changes were prevented by concomitant Transforming Growth Factor β inhibition. Conclusions Our results indicate that chronic TNFα-exposure is sufficient to alter RPE morphology and impede cardinal features that define the differentiated state of RPE cells with striking similarities to the alterations that are observed with age in neurodegenerative diseases such as age-related macular degeneration.
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Chronic exposure to tumor necrosis factor alpha induces retinal pigment epithelium cell dedifferentiation.
Journal of Neuroinflammation, 2018Co-Authors: Sara Touhami, Thibaud Mathis, Fanny Beguier, Sacha Reichman, Sébastien Augustin, Hugo Charles-messance, Lucile Vignaud, Emeline F. Nandrot, Valérie Forster, José-alain SahelAbstract:The retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important barrier and immuno-suppressive functions in the eye. We have previously shown that acute stimulation of RPE cells by tumor necrosis factor alpha (TNFα) downregulates the expression of OTX2 (Orthodenticle homeobox 2) and dependent RPE genes. We here investigated the long-term effects of TNFα on RPE cell morphology and key functions in vitro. Primary porcine RPE cells were exposed to TNFα (at 0.8, 4, or 20 ng/ml per day) for 10 days. RPE cell morphology, phagocytosis, barrier- and immunosuppressive-functions were assessed. Chronic (10 days) exposure of primary RPE cells to TNFα increases RPE cell size and polynucleation, decreases visual cycle gene expression, impedes RPE tight-junction organization and Transepithelial Resistance, and decreases the immunosuppressive capacities of the RPE. TNFα-induced morphological- and Transepithelial-Resistance changes were prevented by concomitant Transforming Growth Factor β inhibition. Our results indicate that chronic TNFα-exposure is sufficient to alter RPE morphology and impede cardinal features that define the differentiated state of RPE cells with striking similarities to the alterations that are observed with age in neurodegenerative diseases such as age-related macular degeneration.
Patricia A Damore - One of the best experts on this subject based on the ideXlab platform.
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oxidized lipoprotein uptake through the cd36 receptor activates the nlrp3 inflammasome in human retinal pigment epithelial cells
Investigative Ophthalmology & Visual Science, 2016Co-Authors: Gopalan Gnanaguru, Ariel R Choi, Dhanesh Amarnani, Patricia A DamoreAbstract:PURPOSE Accumulation of oxidized phospholipids/lipoproteins with age is suggested to contribute to the pathogenesis of AMD. We investigated the effect of oxidized LDL (ox-LDL) on human RPE cells. METHODS Primary human fetal RPE (hf-RPE) and ARPE-19 cells were treated with different doses of LDL or ox-LDL. Assessment of cell death was measured by lactate dehydrogenase release into the conditioned media. Barrier function of RPE was assayed by measuring Transepithelial Resistance. Lysosomal accumulation of ox-LDL was determined by immunostaining. Expression of CD36 was determined by RT-PCR; protein blot and function was examined by receptor blocking. NLRP3 inflammasome activation was assessed by RT-PCR, protein blot, caspase-1 fluorescent probe assay, and inhibitor assays. RESULTS Treatment with ox-LDL, but not LDL, for 48 hours caused significant increase in hf-RPE and ARPE-19 (P < 0.001) cell death. Oxidized LDL treatment of hf-RPE cells resulted in a significant decrease in Transepithelial Resistance (P < 0.001 at 24 hours and P < 0.01 at 48 hours) relative to LDL-treated and control cells. Internalized ox-LDL was targeted to RPE lysosomes. Uptake of ox-LDL but not LDL significantly increased CD36 protein and mRNA levels by more than 2-fold. Reverse transcription PCR, protein blot, and caspase-1 fluorescent probe assay revealed that ox-LDL treatment induced NLRP3 inflammasome when compared with LDL treatment and control. Inhibition of NLRP3 activation using 10 μM isoliquiritigenin significantly (P < 0.001) inhibited ox-LDL induced cytotoxicity. CONCLUSIONS These data are consistent with the concept that ox-LDL play a role in the pathogenesis of AMD by NLRP3 inflammasome activation. Suppression of NLRP3 inflammasome activation could attenuate RPE degeneration and AMD progression.
Sara Touhami - One of the best experts on this subject based on the ideXlab platform.
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Chronic exposure to tumor necrosis factor alpha induces retinal pigment epithelium cell dedifferentiation
Journal of Neuroinflammation, 2018Co-Authors: Sara Touhami, Thibaud Mathis, Fanny Beguier, Sacha Reichman, Sébastien Augustin, Hugo Charles-messance, Lucile Vignaud, Emeline F. Nandrot, Valérie Forster, José-alain SahelAbstract:Background The retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important barrier and immuno-suppressive functions in the eye. We have previously shown that acute stimulation of RPE cells by tumor necrosis factor alpha (TNFα) downregulates the expression of OTX2 (Orthodenticle homeobox 2) and dependent RPE genes. We here investigated the long-term effects of TNFα on RPE cell morphology and key functions in vitro. Methods Primary porcine RPE cells were exposed to TNFα (at 0.8, 4, or 20 ng/ml per day) for 10 days. RPE cell morphology, phagocytosis, barrier- and immunosuppressive-functions were assessed. Results Chronic (10 days) exposure of primary RPE cells to TNFα increases RPE cell size and polynucleation, decreases visual cycle gene expression, impedes RPE tight-junction organization and Transepithelial Resistance, and decreases the immunosuppressive capacities of the RPE. TNFα-induced morphological- and Transepithelial-Resistance changes were prevented by concomitant Transforming Growth Factor β inhibition. Conclusions Our results indicate that chronic TNFα-exposure is sufficient to alter RPE morphology and impede cardinal features that define the differentiated state of RPE cells with striking similarities to the alterations that are observed with age in neurodegenerative diseases such as age-related macular degeneration.
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Chronic exposure to tumor necrosis factor alpha induces retinal pigment epithelium cell dedifferentiation.
Journal of Neuroinflammation, 2018Co-Authors: Sara Touhami, Thibaud Mathis, Fanny Beguier, Sacha Reichman, Sébastien Augustin, Hugo Charles-messance, Lucile Vignaud, Emeline F. Nandrot, Valérie Forster, José-alain SahelAbstract:The retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important barrier and immuno-suppressive functions in the eye. We have previously shown that acute stimulation of RPE cells by tumor necrosis factor alpha (TNFα) downregulates the expression of OTX2 (Orthodenticle homeobox 2) and dependent RPE genes. We here investigated the long-term effects of TNFα on RPE cell morphology and key functions in vitro. Primary porcine RPE cells were exposed to TNFα (at 0.8, 4, or 20 ng/ml per day) for 10 days. RPE cell morphology, phagocytosis, barrier- and immunosuppressive-functions were assessed. Chronic (10 days) exposure of primary RPE cells to TNFα increases RPE cell size and polynucleation, decreases visual cycle gene expression, impedes RPE tight-junction organization and Transepithelial Resistance, and decreases the immunosuppressive capacities of the RPE. TNFα-induced morphological- and Transepithelial-Resistance changes were prevented by concomitant Transforming Growth Factor β inhibition. Our results indicate that chronic TNFα-exposure is sufficient to alter RPE morphology and impede cardinal features that define the differentiated state of RPE cells with striking similarities to the alterations that are observed with age in neurodegenerative diseases such as age-related macular degeneration.
