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Sample records for epithelial cell culture

  1. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  2. Growth requirements of human mammary epithelial cells in culture.

    PubMed

    Taylor-Papadimitriou, J; Shearer, M; Stoker, M G

    1977-12-15

    Colony-forming epithelial cells can be separated from the non-dividing "foam cells" in human milk by differential adhesion to glass and freezing. The growth of such partially purified mammary epithelial cells is stimulated by co-culture with non-dividing feeder cells. Foam cells, mitomycin-treated mouse fibroblast lines and human mammary fibroblasts and calf lens epithelial cells are all effective in promoting mammary epithelial cell growth. Contact between epithelial cells and feeders is not required for the growth-promoting effect. The mitogenic effect of epidermal growth factor on mammary epithelial cells also requires feeder cell activity.

  3. Culture and differentiation of mouse tracheal epithelial cells.

    PubMed

    You, Yingjian; Brody, Steven L

    2013-01-01

    Airway epithelial cell biology has been greatly advanced by studies of genetically defined and modified mice; however it is often difficult to isolate, manipulate, and assay epithelial cell-specific responses in vivo. In vitro proliferation and differentiation of mouse airway epithelial cells are made possible by a high-fidelity system for primary culture of mouse tracheal epithelial cells described in this chapter. Using this method, epithelial cells purified from mouse tracheas proliferate in growth factor-enriched medium. Subsequent culture in defined medium and the use of the air-liquid interface condition result in the development of well-differentiated epithelia composed of ciliated and non-ciliated cells with characteristics of native airways. Methods are also provided for manipulation of differentiation and analysis of differentiation and gene expression. These approaches allow the assessment of global responses and those of specific cell subpopulations within the airway epithelium.

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

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

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

  5. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

    The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. Images PMID:6933523

  6. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-01-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips, including methods to perform electrical impedance spectroscopy, methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry, techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress, and methods to carry out high-resolution imaging of vesicular trafficking with light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  7. Lingual Epithelial Stem Cells and Organoid Culture of Them

    PubMed Central

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-01

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine. PMID:26828484

  8. Type II alveolar epithelial cell in vitro culture in aerobiosis.

    PubMed

    Aerts, C; Voisin, C; Wallaert, B

    1988-08-01

    A method of Type II alveolar epithelial cell culture in aerobiosis has been developed. Isolation of Type II cells was performed by digesting guinea-pig lung tissue with crude trypsin and elastase and using discontinuous Percoll density gradients. The Type II cells, as identified by light and electron microscopy, were cultured in aerobiosis for up to six days, in direct contact with the atmosphere in conditions mimicking those present in the lower respiratory tract. Significant activities of cellular superoxide dismutase (SOD), manganese dependent superoxide dismutase (Mn-SOD), catalase and glutathione peroxidase (GSH-Px) were found at the time of isolation. In contrast, cell glutathione content varied widely from one experiment to another. Changes of antioxidant enzymes were evaluated during cell culture in aerobiosis. SOD, Mn-SOD and catalase were significantly decreased after three days but were not significantly different between a three day and six day culture. Antioxidant changes did not influence the cell culture. In marked contrast, decrease in cell glutathione was associated with rapid cell death, whereas good cell survival was obtained at high levels of cell glutathione. Cell culture in aerobiosis will permit a precise evaluation of the effects of gases, particularly oxidant gases, on a primary culture of Type II alveolar epithelial cells.

  9. Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Kikuchi, Tetsutaro; Kitano, Yuriko; Watanabe, Hiroya; Mizutani, Manabu; Nozaki, Takayuki; Senda, Naoko; Saitoh, Kazuo; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-05-01

    Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets.

  10. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

  11. In vitro methods to culture primary human breast epithelial cells.

    PubMed

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  12. Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells.

    PubMed

    Takano, Mikihisa; Nagahiro, Machi; Yumoto, Ryoko

    2016-02-01

    Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation.

  13. Differentiation of cultured epithelial cells: response to toxic agents.

    PubMed Central

    Rice, R H; LaMontagne, A D; Petito, C T; Rong, X H

    1989-01-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Great differences were evident even among those cells derived from stratified squamous epithelia (epidermal, esophageal, vaginal, forestomach) despite their expression of aryl hydrocarbon hydroxylase activities to similar degrees. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAMP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Although expressing keratinocyte character (transglutaminase activity and envelope forming ability), the cells thus retain some hormonal character that may be modulated by cAMP-dependent kinase activity. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents. Images FIGURE 1. FIGURE 4. PMID:2466642

  14. Three-Dimensional Cultures of Mouse Mammary Epithelial Cells

    PubMed Central

    Mroue, Rana; Bissell, Mina J.

    2013-01-01

    The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our

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

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  16. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model

    EPA Science Inventory

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  17. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    SciTech Connect

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  18. Culture models of human mammary epithelial cell transformation

    SciTech Connect

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  19. Epithelial-mesenchymal transitions during cell culture of primary thyroid tumors?

    PubMed

    Herrmann, M E; Trevor, K T

    1993-04-01

    Fibroblast contamination of epithelial tumor cell cultures is of great concern when examining tumor cells in vitro for specific biochemical and cytogenetic changes. The observations of normal karyotypes in thyroid tumor cell cultures have raised the concern of whether residual tissue fibroblasts might obscure the cytogenetic analysis of transformed epithelial cells. We have characterized early passaged thyroid tumor cells to examine the proportions of epithelial and fibroblastic cell types. Cells were analyzed by immunocytology using antibodies recognizing the thyroid prohormone thyroglobulin, epithelial cytokeratins, and vimentin, a mesenchyme marker. Tumors consisted of one follicular adenoma and five papillary carcinomas. When examined by day 15 in culture, all cells contained filaments composed of vimentin, which most likely represents an adaptation to culture conditions. Double immunofluorescence staining for thyroglobulin and cytokeratin revealed the presence of not only epithelial but also spindle-like fibroblastoid cells possessing thyroid epithelial cell markers. The results suggest that in thyroid tumor cultures there is a unique cell type intermediate between epithelial and mesenchyme phenotypes that must be considered when performing cytogenetic analysis.

  20. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    PubMed

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  1. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

  2. Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture

    PubMed Central

    Gidfar, Sanaz; Milani, Farnoud Y.; Milani, Behrad Y.; Shen, Xiang; Eslani, Medi; Putra, Ilham; Huvard, Michael J.; Sagha, Hossein; Djalilian, Ali R.

    2017-01-01

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells. PMID:28054657

  3. Nerve growth factor modulate proliferation of cultured rabbit corneal endothelial cells and epithelial cells.

    PubMed

    Li, Xinyu; Li, Zhongguo; Qiu, Liangxiu; Zhao, Changsong; Hu, Zhulin

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  4. Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro.

    PubMed

    Jiao, Jian; Meng, Na; Wang, Hong; Zhang, Luo

    2013-12-01

    The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IVand ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a highspeed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th-10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78 ± 1.99 Hz and 7.91 ± 2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

  5. Epithelial monolayer culture system for real‐time single‐cell analyses

    PubMed Central

    Seo, Jong Bae; Moody, Mark; Koh, Duk‐Su

    2014-01-01

    Abstract Many epithelial cells form polarized monolayers under in vivo and in vitro conditions. Typically, epithelial cells are cultured for differentiation on insert systems where cells are plated on a porous filter membrane. Although the cultured monolayers have been a standard system to study epithelial physiology, there are some limits: The epithelial cells growing inside the commercial inserts are not optimal to visualize directly through lenses on inverted microscopes. The cell images are optically distorted and background fluorescence is bright due to the filter membrane positioned between the cells and the lens. In addition, the cells are not easily accessible by electrodes due to the presence of tall side walls. Here, we present the design, fabrication, and practical applications of an improved system for analysis of polarized epithelial monolayers. This new system allows (1) direct imaging of cells without an interfering filter membrane, (2) electrophysiological measurements, and (3) detection of apical secretion with minimal dilution. Therefore, our culture method is optimized to study differentiated epithelial cells at the single‐cell and subcellular levels, and can be extended to other cell types with minor modifications. PMID:24771696

  6. Culture of Airway Epithelial Cells from Neonates Sampled within 48-Hours of Birth

    PubMed Central

    Miller, David; Turner, Steve W.; Spiteri-Cornish, Daniella; McInnes, Neil; Scaife, Alison; Danielian, Peter J.; Devereux, Graham; Walsh, Garry M.

    2013-01-01

    Introduction Little is known about how neonatal airway epithelial cell phenotype impacts on respiratory disease in later life. This study aimed to establish a methodology to culture and characterise neonatal nasal epithelial cells sampled from healthy, non-sedated infants within 48 hours of delivery. Methods Nasal epithelial cells were sampled by brushing both nostrils with an interdental brush, grown to confluence and sub-cultured. Cultured cells were characterised morphologically by light and electron microscopy and by immunocytochemistry. As an exemplar pro-inflammatory chemokine, IL-8 concentrations were measured in supernatants from unstimulated monolayers and after exposure to IL-1β/TNF-α or house dust mite extract. Results Primary cultures were successfully established in 135 (91%) of 149 neonatal samples seeded, with 79% (n  =  117) successfully cultured to passage 3. The epithelial lineage of the cells was confirmed by morphological analysis and immunostaining. Constitutive IL-8 secretion was observed and was upregulated by IL-1β/TNF-α or house dust mite extract in a dose dependent manner. Conclusion We describe a safe, minimally invasive method of culturing nasal epithelial cells from neonates suitable for functional cell analysis offering an opportunity to study “naïve” cells that may prove useful in elucidating the role of the epithelium in the early origins of asthma and/or allergic rhinitis. PMID:24223790

  7. Isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture.

    PubMed

    Xu, Kun; Buchsbaum, Rachel J

    2012-04-30

    While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors(1-2), much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension(3-4). Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture(3). While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis. The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized(4). Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells(5). Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals(6). Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors(7). Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion(8-15). We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three

  8. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells

    PubMed Central

    Drummond, Coyne G.

    2015-01-01

    ABSTRACT Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and

  9. Organotypic culture of fetal lung type II alveolar epithelial cells: applications to pulmonary toxicology.

    PubMed Central

    Shami, S G; Aghajanian, J D; Sanders, R L

    1984-01-01

    Techniques for isolation and culture of fetal Type II alveolar epithelial cells, as well as the morphologic and biochemical characteristics of these histotypic cultures, are described. Type II alveolar epithelial cells can be isolated from fetal rat lungs and grown in an organotypic culture system as described in this review. The fetal Type II cells resemble differentiated rat Type II cells in morphology, biochemistry, and karyotype as they grow in culture for up to 5 weeks. The cells of the mature organotypic cultures form alveolarlike structures while growing on a gelatin sponge matrix. The Type II cells also synthesize and secrete pulmonary surfactant similar in biochemical composition to that produced in vivo. This system has been used to study the effects of hormones on surfactant production and composition. The organotypic model has many potential applications to the study of pulmonary toxicology. Images FIGURE 1. FIGURE 2. PMID:6548184

  10. CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

  11. Effect of cord blood serum on ex vivo human limbal epithelial cell culture.

    PubMed

    Chakraborty, Anindita; Dutta, Jayanta; Das, Sumantra; Datta, Himadri

    2012-12-01

    Limbal cell transplantation is an efficacious procedure for rehabilitation of visual acuity in patients with severe ocular surface disorders. Cultivation of limbal epithelial stem cell with fetal bovine serum for transplantation has been a promising treatment for reconstructing the ocular surface in severe limbal stem cell deficiency caused by Steven Johnson syndrome, chemical or thermal injury. This technique of "cell therapy" has been accepted worldwide but the cost of cultivating the cells for transplantation is high. The objective of this study was to investigate the effect of cord blood serum in place of fetal bovine serum on the growth of human limbal epithelial cell culture. Our group has experimented with human cord blood serum which was obtained free of cost from willing donors. The use of human cord blood serum in place of fetal bovine serum for ex vivo culture of limbal stem cell has helped us in reducing the cost of culture. Fresh human limbal tissues from donor cadavers were cultured on intact and denuded amniotic membrane. Cells were proliferated in vitro with cell culture media containing human cord blood serum. Reverse transcription-polymerase chain reaction and immunofluorescence cytochemistry of cultured human limbal epithelial stem cell was done for characterization of the cells.

  12. Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid.

    PubMed

    Basso, Fernanda Gonçalves; Soares, Diana Gabriela; Pansani, Taisa Nogueira; Turrioni, Ana Paula Silveira; Scheffel, Débora Lopes; Hebling, Josimeri; Costa, Carlos Alberto de Souza

    2016-11-28

    Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA) on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 μM) for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

  13. An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

    PubMed Central

    Birch, Nigel P.; Suresh, Vinod

    2016-01-01

    Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10−8 cm/s vs (738 ± 190) ×10−8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI

  14. Neurons Self-Organize Around Salivary Epithelial Cells in Novel Co-Culture Model

    PubMed Central

    Sommakia, Salah; Baker, Olga J.

    2016-01-01

    Salivary gland bioengineering requires understanding the interaction between salivary epithelium and surrounding tissues. An important component of salivary glands is the presence of neurons. No previous studies have investigated how neurons and salivary epithelial cells interact in an in vitro co-culture model. In this study, we describe the self-organization of neurons around salivary epithelial cells in co-culture, in a similar fashion to what occurs in native tissue. We cultured primary mouse cortical neurons (m-CN) with a salivary epithelial cell line (Par-C10) on growth factor-reduced Matrigel (GFR-MG) for 4 days. After this time, co-cultures were compared with native salivary glands using confocal microscopy. Our findings indicate that m-CN were able to self-organize basolaterally to salivary epithelial cell clusters in a similar manner to what occurs in native tissue. These results indicate that this model can be developed as a potential platform for studying neuron-salivary epithelial cell interactions for bioengineering purposes. PMID:27833941

  15. Cytoskeleton remodelling of confluent epithelial cells cultured on porous substrates

    PubMed Central

    Rother, Jan; Büchsenschütz-Göbeler, Matthias; Nöding, Helen; Steltenkamp, Siegfried; Samwer, Konrad; Janshoff, Andreas

    2015-01-01

    The impact of substrate topography on the morphological and mechanical properties of confluent MDCK-II cells cultured on porous substrates was scrutinized by means of various imaging techniques as well as atomic force microscopy comprising force volume and microrheology measurements. Regardless of the pore size, ranging from 450 to 5500 nm in diameter, cells were able to span the pores. They did not crawl into the holes or grow around the pores. Generally, we found that cells cultured on non-porous surfaces are stiffer, i.e. cortical tension rises from 0.1 to 0.3 mN m−1, and less fluid than cells grown over pores. The mechanical data are corroborated by electron microscopy imaging showing more cytoskeletal filaments on flat samples in comparison to porous ones. By contrast, cellular compliance increases with pore size and cells display a more fluid-like behaviour on larger pores. Interestingly, cells on pores larger than 3500 nm produce thick actin bundles that bridge the pores and thereby strengthen the contact zone of the cells. PMID:25566882

  16. Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

    NASA Technical Reports Server (NTRS)

    Richmond, Robert C.

    2001-01-01

    Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

  17. A differentiated porcine bronchial epithelial cell culture model for studying human adenovirus tropism and virulence.

    PubMed

    Lam, E; Ramke, M; Groos, S; Warnecke, G; Heim, A

    2011-12-01

    The species specificity of human adenoviruses (HAdV) almost precludes studying virulence and tropism in animal models, e.g. rodent models, or derived tissue and cell culture models. However, replication of HAdV type 5 (HAdV-C5) has been shown after intravenous injection in swine. In order to study adenovirus replication in airway tissue propagation of bronchial epithelial cells from porcine lungs was established. These primary cells proved to be fully permissive for HAdV-C5 infection in submerged culture, demonstrating efficient HAdV genome replication, infectious viral particle release (1.07×10(8) TCID(50)/ml±6.63×10(7)) and development of cytopathic effect (CPE). Differentiation of porcine bronchial epithelial cells was achieved at the air-liquid interface on collagen I coated 0.4μm polyester membranes. Morphology, expression of tubulin and occludin, the development of tight-junctions and cilia were similar to human bronchial epithelial cells. Infection with HAdV-C5 from the basolateral side resulted in release of infectious virus progeny (2.05×10(7) TCID(50)/ml±2.39×10(7)) to the apical surface as described recently in human bronchial epithelial cells, although complete CPE was not observed. Differentiated porcine bronchial epithelial cells hold promise as a novel method for studying the virulence and pathophysiology of pneumonia associated HAdV types.

  18. Estrogen inhibits cell cycle progression and retinoblastoma phosphorylation in rhesus ovarian surface epithelial cell culture

    SciTech Connect

    Wright, Jay W.; Stouffer, Richard L.; Rodland, Karin D.

    2003-10-31

    Estrogen promotes the growth of some ovarian cancer cells at nanomolar concentrations, but has been shown to inhibit growth of normal ovarian surface epithelial (OSE) cells at micromolar concentrations (1μg/ml). OSE cells express the estrogen receptor (ER)-α, and are the source of 90% of various cancers. The potential sensitivity of OSE cells to estrogen stresses the importance of understanding the estrogen-dependent mechanisms at play in OSE proliferation and transformation, as well as in anticancer treatment. We investigated the effects of estradiol on cell proliferation in vitro, and demonstrate an intracellular locus of action of estradiol in cultured rhesus ovarian surface epithelial (RhOSE) cells. We show that ovarian and breast cells are growth-inhibited by micromolar concentration of estradiol and that this inhibition correlates with estrogen receptor expression. We further show that normal rhesus OSE cells do not activate ERK or Akt in response to estradiol nor does estradiol block the ability of serum to stimulate ERK or induce cyclin D expression. Contrarily, estradiol inhibits serum-dependent retinoblastoma protein (Rb) phosphorylation and blocks DNA synthesis. This inhibition does not formally arrest cells and is reversible within hours of estrogen withdrawal. Our data are consistent with growth inhibition by activation of Rb and indicate that sensitivity to hormone therapy in anticancer treatment can be modulated by cell cycle regulators downstream of the estrogen receptor.

  19. IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus.

    PubMed

    Vilela, Ricardo Chaves; Benchimol, Marlene

    2013-02-01

    Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

  20. Bovine oviductal epithelial cells: long term culture characterization and impact of insulin on cell morphology.

    PubMed

    Palma-Vera, S; Einspanier, R; Schoen, J

    2014-09-01

    In vitro models that resemble cell function in vivo are needed to understand oviduct physiology. This study aimed to assess cell functions and insulin effects on bovine oviductal epithelial cells (BOECs) cultured in an air-liquid interface. BOECs (n=6) were grown in conditioned Ham's F12, DMEM or Ham's F12/DMEM with 10% fetal calf serum (FCS) for 3 weeks. After selecting the most suitable medium (Ham's F12), increasing insulin concentrations (1 ng/mL, 20 ng/mL and 5 μg/mL) were applied, and cell morphology and trans-epithelial electrical resistance (TEER; n=4) were evaluated after 3 and 6 weeks. Keratin immunohistochemistry and mRNA expression of oviductal glycoprotein 1 (OVGP1) and progesterone receptor (PGR) were conducted (n=4) to assess cell differentiation. BOECs grown without insulin supplementation or with 1 ng/mL of insulin displayed polarization and secretory activity. However, cells exhibited only 50% of the height of their in vivo counterparts. Cultures supplemented with 20 ng/mL insulin showed the highest quality, but the 5 μg/mL concentration induced massive growth. TEER correlated negatively with insulin concentration (r=-0.459; p=0.009). OVGP1 and PGR transcripts were still detectable after 3 and 6 weeks. Cellular localization of keratins closely resembled that of BOECs in vivo. Cultures showed heterogeneous expression of PGR and OVGP1 in response to estradiol (10 pg/mL). In summary, BOECs grown for long term in an air-liquid interface expressed markers of cell differentiation. Additionally, insulin supplementation (20 ng/mL) improved the cell morphology in vitro.

  1. Establishment of three-dimensional cultures of human pancreatic duct epithelial cells

    SciTech Connect

    Gutierrez-Barrera, Angelica M.; Menter, David G.; Abbruzzese, James L.; Reddy, Shrikanth A.G. . E-mail: sa08366@wotan.mdacc.tmc.edu

    2007-07-06

    Three-dimensional (3D) cultures of epithelial cells offer singular advantages for studies of morphogenesis or the role of cancer genes in oncogenesis. In this study, as part of establishing a 3D culture system of pancreatic duct epithelial cells, we compared human pancreatic duct epithelial cells (HPDE-E6E7) with pancreatic cancer cell lines. Our results show, that in contrast to cancer cells, HPDE-E6E7 organized into spheroids with what appeared to be apical and basal membranes and a luminal space. Immunostaining experiments indicated that protein kinase Akt was phosphorylated (Ser473) and CTMP, a negative Akt regulator, was expressed in both HPDE-E6E7 and cancer cells. However, a nuclear pool of CTMP was detectable in HPDE-E6E7 cells that showed a dynamic concentrated expression pattern, a feature that further distinguished HPDE-E637 cells from cancer cells. Collectively, these data suggest that 3D cultures of HPDE-E6E7 cells are useful for investigating signaling and morphological abnormalities in pancreatic cancer cells.

  2. Long-term culture and partial characterization of dog gallbladder epithelial cells

    SciTech Connect

    Oda, D.; Lee, S.P.; Hayashi, A. )

    1991-05-01

    We describe the successful isolation and maintenance of primary cultures of dog gallbladder epithelial cells. The surgically removed gallbladder was treated with trypsin/EDTA for 45 minutes and epithelial cells were collected and resuspended in Eagle's minimum essential medium with 10% fetal calf serum, and plated on Vitrogen-coated culture dishes. Each gallbladder yielded approximately 12 to 15 x 10{sup 6} columnar epithelial cells, greater than 95% of which were viable by trypan blue exclusion. In culture, cells maintained their polarity. They were arranged and grew in small and tight clusters that coalesced at confluency. When examined using transmission electron microscopy, prominent and numerous microville were identified on the apical portion of the plasma membrane. Cells were connected by well-formed desmosomes. Scanning electron microscopy revealed clusters of polyhedral cells with numerous papillary projections. Immunohistochemical studies demonstrated uniform staining of cells to keratin 35BH11 and AE1. Histochemical studies were positive for gamma-glutamyl transpeptidase and negative for glucose-6-phosphatase and albumin. Cells incorporated ({sup 3}H)uridine into intracellular proteins and ({sup 14}C)glucosamine into tissue and secreted mucous glycoproteins linearly over 2 to 24 hours. Flow cytometry studies demonstrated a consistent and reproducible number of cells (10 to 12%) at S-phase. However, the number of cells at S-phase was dramatically reduced to almost negligible as cells reached confluency. This method of culturing primary dog gallbladder epithelial cells is highly reproducible and reliable. These cells preserve their state of differentiation, polarity, histochemical and immunohistochemical profile, morphologic, and metabolic integrity with repeated passaging or after being frozen.

  3. Ultrasonic differentiation of normal versus malignant breast epithelial cells in monolayer cultures

    PubMed Central

    Doyle, Timothy E.; Goodrich, Jeffrey B.; Ambrose, Brady J.; Patel, Hemang; Kwon, Soonjo; Pearson, Lee H.

    2010-01-01

    Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different. PMID:21110531

  4. Enhanced growth medium and method for culturing human mammary epithelial cells

    DOEpatents

    Stampfer, Martha R.; Smith, Helene S.; Hackett, Adeline J.

    1983-01-01

    Methods are disclosed for isolating and culturing human mammary epithelial cells of both normal and malignant origin. Tissue samples are digested with a mixture including the enzymes collagenase and hyaluronidase to produce clumps of cells substantially free from stroma and other undesired cellular material. Growing the clumps of cells in mass culture in an enriched medium containing particular growth factors allows for active cell proliferation and subculture. Clonal culture having plating efficiencies of up to 40% or greater may be obtained using individual cells derived from the mass culture by plating the cells on appropriate substrates in the enriched media. The clonal growth of cells so obtained is suitable for a quantitative assessment of the cytotoxicity of particular treatment. An exemplary assay for assessing the cytotoxicity of the drug adriamycin is presented.

  5. Stromal-epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model.

    PubMed

    Kobayashi, Takeshi; Shiraishi, Atsushi; Hara, Yuko; Kadota, Yuko; Yang, Lujun; Inoue, Tomoyuki; Shirakata, Yuji; Ohashi, Yuichi

    2015-06-01

    Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal-epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal-epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin-eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two-three layers after five days and five-seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-β1, TGF-β2, and TGF-β3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-β family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal-epithelial cell

  6. MCP-1 expression by rat type II alveolar epithelial cells in primary culture.

    PubMed

    Paine, R; Rolfe, M W; Standiford, T J; Burdick, M D; Rollins, B J; Strieter, R M

    1993-05-15

    Recruitment and activation of mononuclear phagocytes are potentially critical regulatory events for control of pulmonary inflammation. Located at the boundary between the alveolar airspace and the interstitium, alveolar epithelial cells are ideally situated to regulate the recruitment and activation of mononuclear phagocytes through the production of cytokines in response to inflammatory stimulation from the alveolar space. To test this hypothesis, we investigated the production of monocyte chemotactic polypeptide-1 (MCP-1), a protein that is chemotactic for and that activates monocytes, by rat type II alveolar epithelial cells in primary culture. Immunocytochemical staining using anti-murine JE, an antibody recognizing rat MCP-1, demonstrated cell-associated MCP-1 Ag throughout the monolayer. The intensity of staining was increased in response to IL-1 beta. When type II epithelial cells formed a tight monolayer on a filter support, there was polar secretion of MCP-1 Ag into the apical compartment by both control and IL-1-stimulated cells as measured by specific MCP-1 ELISA. Northern blot analysis revealed that IL-1 and TNF-alpha stimulated MCP-1 mRNA expression in a dose-dependent manner, whereas dexamethasone blocked MCP-1 expression by cells stimulated with IL-1. In contrast to previous results using transformed epithelial cell lines, MCP-1 mRNA was induced in these primary cultures directly by stimulation with LPS. These data suggest that alveolar epithelial cells may have an important and previously unrecognized role in the initiation and maintenance of inflammatory processes in the lung by recruiting and activating circulating monocytes through the production of MCP-1.

  7. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    PubMed

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  8. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations

    PubMed Central

    Fronk, Aaron H; Vargis, Elizabeth

    2016-01-01

    The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included. PMID:27493715

  9. Autologous Transplantation of Oral Mucosal Epithelial Cell Sheets Cultured on an Amniotic Membrane Substrate for Intraoral Mucosal Defects

    PubMed Central

    Amemiya, Takeshi; Nakamura, Takahiro; Yamamoto, Toshiro; Kinoshita, Shigeru; Kanamura, Narisato

    2015-01-01

    The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects. PMID:25915046

  10. The biomedical aspects of oral mucosal epithelial cell culture in mammals.

    PubMed

    Bryja, A; Dyszkiewicz-Konwińska, M; Budna, J; Kranc, W; Chachuła, A; Borys, S; Ciesiółka, S; Sokalski, J; Prylinski, M; Bukowska, D; Antosik, P; Bruska, M; Nowicki, M; Zabel, M; Kempisty, B

    2017-01-01

    In recent years, there has been a growing interest in epithelial cell tissue culture, particularly oral mucosa and its application utilizing in vitro cell culture in medicine. This involves tests using animal models to better understand oral mucosa function, and the differences in its construction in various animal models. The use of buccal pouch mucosal cell culture provides insight into the processes of trans mucosal transport and regeneration of the oral epithelium. The processes associated with epithelium regeneration is the base for stem cell research and/or oral cancer investigation. These artificially cultured tissue equivalents are used in transplant surgery for the treatment of a variety of tissue dysfunctions, i.e. eye, esophagus, or urethra. In this review, the most recent results from studies carried out on in animal models, which may be applied in areas such as regenerative medicine and reconstructive surgery, were explored.

  11. Corneal regeneration by transplantation of corneal epithelial cell sheets fabricated with automated cell culture system in rabbit model.

    PubMed

    Kobayashi, Toyoshige; Kan, Kazutoshi; Nishida, Kohji; Yamato, Masayuki; Okano, Teruo

    2013-12-01

    We have performed clinical applications of cell sheet-based regenerative medicine with human patients in several fields. In order to achieve the mass production of transplantable cell sheets, we have developed automated cell culture systems. Here, we report an automated robotic system utilizing a cell culture vessel, cell cartridge. The cell cartridge had two rooms for epithelial cells and feeder layer cells separating by porous membrane on which a temperature-responsive polymer was covalently immobilized. After pouring cells into this robotic system, cell seeding, medium change, and microscopic examination during culture were automatically performed according to the computer program. Transplantable corneal epithelial cell sheets were successfully fabricated in cell cartridges with this robotic system. Then, fabricated cell sheets were transplanted onto ocular surfaces of rabbit limbal epithelial stem cell deficiency model after 6-h transportation using a portable homothermal container to keep inner temperature at 36 °C. Within one week after transplantation, normal corneal epithelium was successfully regenerated. This automatic cell culture system would be useful for industrialization of tissue-engineered products for regenerative medicine.

  12. Bovine mammary epithelial cells retain stem-like phenotype in long-term cultures.

    PubMed

    Cravero, Diego; Diego, Cravero; Martignani, Eugenio; Eugenio, Martignani; Miretti, Silvia; Silvia, Miretti; Macchi, Elisabetta; Elisabetta, Macchi; Accornero, Paolo; Paolo, Accornero; Baratta, Mario; Mario, Baratta

    2014-10-01

    The detection and characterization of bovine mammary stem cells may give a better understanding of the cyclic characteristic of mammary gland development. In turn, this could potentially offer techniques to manipulate lactation yield and for regenerative medicine. We previously demonstrated that adult stem cells reside in the bovine mammary gland and possess an intrinsic regenerative potential. In vitro maintenance and expansion of this primitive population is a challenging task that could make easier the study of adult mammary stem cells. The aim of this study is to investigate this possibility. Different subpopulations of mammary epithelial cells emerge when they are cultured in two defined culture conditions. Specific cell differentiation markers as cytokeratin 18 (CK18) and cytokeratin 14 (CK14) were expressed with significant differences according to culture conditions. Vimentin, a well-known fibroblast marker was observed to increase significantly (P < 0.5) only after day 20. In both conditions, after prolonged culture (25 days) a subset of cells still retained regenerative capabilities. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice as shown by the expression of cytokeratin 14 (CK14), cytokeratin 18 (CK18), p63 (a mammary basal cell layer marker) and Epithelial Cell Adhesion Molecule (EpCAM). We also were able to observe the presence of milk proteins signal in these regenerated structures, which is a specific marker of functional mammary alveoli. Progenitor content was also analyzed in vitro through Colony-Forming Cell (CFC) assays with no substantial differences among culture conditions and time points. These results demonstrate that long-term culture of a multipotent cell subpopulation with intrinsic regenerative potential is possible.

  13. Organotypic culture in three dimensions prevents radiation-induced transformation in human lung epithelial cells

    PubMed Central

    El-Ashmawy, Mariam; Coquelin, Melissa; Luitel, Krishna; Batten, Kimberly; Shay, Jerry W.

    2016-01-01

    The effects of radiation in two-dimensional (2D) cell culture conditions may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture. In this study, we determined if the frequency of radiation-induced transformation and cancer progression differed in 3D compared to 2D culture. Telomerase immortalized human bronchial epithelial cells (HBECs) with shTP53 and mutant KRas expression were exposed to various types of radiation (gamma, +H, 56Fe) in either 2D or 3D culture. After irradiation, 3D structures were dissociated and passaged as a monolayer followed by measurement of transformation, cell growth and expression analysis. Cells irradiated in 3D produced significantly fewer and smaller colonies in soft agar than their 2D-irradiated counterparts (gamma P = 0.0004; +H P = 0.049; 56Fe P < 0.0001). The cell culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation—implying there was no selection against cells in or dissociated from 3D conditions. However, DNA damage repair and apoptosis markers were increased in 2D cells compared to 3D cells after radiation. Ideally, expanding the utility of 3D culture will allow for a better understanding of the biological consequences of radiation exposure. PMID:27539227

  14. Organotypic culture in three dimensions prevents radiation-induced transformation in human lung epithelial cells

    NASA Astrophysics Data System (ADS)

    El-Ashmawy, Mariam; Coquelin, Melissa; Luitel, Krishna; Batten, Kimberly; Shay, Jerry W.

    2016-08-01

    The effects of radiation in two-dimensional (2D) cell culture conditions may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture. In this study, we determined if the frequency of radiation-induced transformation and cancer progression differed in 3D compared to 2D culture. Telomerase immortalized human bronchial epithelial cells (HBECs) with shTP53 and mutant KRas expression were exposed to various types of radiation (gamma, +H, 56Fe) in either 2D or 3D culture. After irradiation, 3D structures were dissociated and passaged as a monolayer followed by measurement of transformation, cell growth and expression analysis. Cells irradiated in 3D produced significantly fewer and smaller colonies in soft agar than their 2D-irradiated counterparts (gamma P = 0.0004 +H P = 0.049 56Fe P < 0.0001). The cell culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation—implying there was no selection against cells in or dissociated from 3D conditions. However, DNA damage repair and apoptosis markers were increased in 2D cells compared to 3D cells after radiation. Ideally, expanding the utility of 3D culture will allow for a better understanding of the biological consequences of radiation exposure.

  15. Splitting culture medium by air-jet and rewetting for the assessment of the wettability of cultured epithelial cell surfaces.

    PubMed

    Tanaka, Nobuyuki; Kondo, Makoto; Uchida, Ryohei; Kaneko, Makoto; Sugiyama, Hiroaki; Yamato, Masayuki; Okano, Teruo

    2013-12-01

    This study found that the phenomenon of rewetting after squeezing culture medium varied in different culture conditions for rat oral mucosal epithelial cells. When culture medium covering over cultured cells was squeezed by an air-jet application, the motion of squeezed culture medium was able to be observed by using a commercially available movie camera. Squeezed width on cells cultured in keratinocyte culture medium (KCM), which contained with fetal bovine serum, was one-sixth of that in FBS-free KCM. This result corresponded to the mucous layer staining statuses of cultured cells in both cases; positive in KCM and negative in FBS-free medium. Furthermore, the gene expression of mucous glycoprotein MUC4 in KCM was 100 times higher than that in FBS-free medium, and the expression of MUC4 protein only showed on the apical surface of cells cultured in KCM. The relative gene expression levels of MUC1, 13, 15, and 16 in both the normal and FBS-free medium were found to be no more than one-thirtieth of that of MUC4 in KCM. The main factor of the wettability difference between KCM and FBS-free medium was speculated to be the difference of MUC4 expression between both media. This method can be a simple technique for testing not only the surface wettability but also the mucous formation of cultured cells.

  16. Effects of steroids on the secretion of immunoregulatory factors by thymic epithelial cell cultures.

    PubMed Central

    Stimson, W H; Crilly, P J

    1981-01-01

    Rat thymic epithelial cells were cultured for 39 days in the presence of various concentrations of oestradiol, testosterone, progesterone and corticosterone and the supernatants assessed for effects on the stimulation of cells from the thymus, bone marrow, lymph nodes and spleen, with several agents. All the steroids, except progesterone, were found to significantly regulate the secretion of immunoregulatory factors by the epithelial cells at physiological levels but the effects were dose dependent. Fractionation of active supernatants indicated that the capacity to enhance or depress cellular proliferation was mainly associated with substances having molecular weights greater than 30,000 or less than 1000, respectively. This study supports the idea that certain steroids can influence the immune response indirectly through the thymus. PMID:7298074

  17. Lens and cataract: clastogenic responses in epithelial cells of the organ-cultured rat lens

    SciTech Connect

    Geard, C.R.; Worgul, B.V.

    1987-01-01

    The epithelial cells of the vertebrate lens have an unique character and a probable involvement in cataract formation, which could be initiated by exogenous stimuli. Individual rat lenses were organ-cultured, and the effects of mitomycin C and gamma rays on sister chromatid exchanges (SCE), chromosomal aberrations, and cellular kinetics assessed in cells from the epithelial monolayer. SCE showed about a 5.5-fold increase over the mitomycin C dose range (0, 17, 83, 170 nM), while chromosomal aberrations increased 38-fold. In cells from untreated lenses, SCE were 1600 times more frequent than aberrations and at a level consistent with in vivo assessments in other cell types. Gamma rays (up to 4 Gy) had a greater inhibiting effect on cellular progression, while 17 nM mitomycin C and 1 Gy induced similar clastogenic responses. This first demonstration of such changes in lens epithelial cells expands on the cell types available for monitoring potential mutagen-carcinogens. Additionally chromosomal changes resulting from lens cellular challenge could be the basis of later cytopathological changes in the lens, of which cataract is the primary concern to humans. Potential cataractogens warrant monitoring, and the study outlined may aid in this endeavor, as well as contributing to an understanding of cataract etiology.

  18. Factors affecting morphogenesis of rabbit gallbladder epithelial cells cultured in collagen gels.

    PubMed

    Mori, M; Miyazaki, K

    2000-05-01

    Although peptide growth factors play an important role in the morphogenesis of gallbladder, little is known about how they effect the morphogenesis of gallbladder epithelial cells. Rabbit gallbladder epithelial cells (RGEC) were isolated and cultured in monolayer or collagen gels. Epidermal growth factor (EGF), hepatocyte growth factor (HGF), epimorphin, transforming growth factor-beta 1 (TGF-beta 1), and fibroblast-conditioned medium (FCM) were added to the cultured cells to clarify the effects of these peptides and FCM on morphogenesis of RGEC. RGEC suspended in collagen gels form spherical cysts with morphologic polarity. EGF, HGF, epimorphin, and FCM promoted cyst maturation by accelerating the proliferation and aggregation of clear, polarized vesicles. In contrast, TGF-beta 1 markedly inhibited DNA synthesis in both monolayer and collagen gel cultures and promoted formation of branching structures in collagen gels. Furthermore, in the presence of EGF, TGF-beta 1 induced a drastic change in morphogenesis, with the formation of branching networks that showed cell-cell contact only at sites where branches touched. RGEC-forming multicellular cysts did not express vimentin but expressed significant amounts of cytokeratin and regained junctional complexes. In contrast, TGF-beta 1-treated cells strongly expressed vimentin along with branching structures and showed decreases in cytokeratin expression and junctional complexes. Thus, TGF-beta 1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular changes, with loss of both epithelial polarization and junctional complexes. These results suggest that the morphogenetic program of RGEC is likely to be determined by the interaction of these peptides and the timing of their presence.

  19. Morphogenetic Effects of Neuregulin (Neu Differentiation Factor) in Cultured Epithelial Cells

    PubMed Central

    Chausovsky, Alexander; Tsarfaty, Ilan; Kam, Zvi; Yarden, Yosef; Geiger, Benjamin; Bershadsky, Alexander D.

    1998-01-01

    Neuregulin, or neu differentiation factor, induces cell proliferation or differentiation through interaction with members of the ErbB family of receptor tyrosine kinases. We report that neuregulin can also induce profound morphogenic responses in cultured epithelial cells of different origins. These effects include scattering of small epithelial islands and rearrangement of larger cell islands into ordered ring-shaped arrays with internal lumens. The ring-forming cells are interconnected by cadherin- and β-catenin-containing adherens junctions. In confluent cultures, neuregulin treatment induces formation of circular lumenlike gaps in the monolayer. Both cell scattering and ring formation are accompanied by a marked increase in cell motility that is independent of hepatocyte growth factor/scatter factor and its receptor (c-Met). Affinity-labeling experiments implied that a combination of ErbB-2 with ErbB-3 mediates the morphogenic signal of neuregulin in gastric cells. Indeed, a similar morphogenic effect could be reconstituted in nonresponsive cells by coexpression of ErbB-2 and -3. We conclude that a heterodimer between the kinase-defective neuregulin receptor, ErbB-3, and the coreceptor, ErbB-2, mediates the morphogenetic action of neuregulin. PMID:9802906

  20. Oxysterols from human bile induce apoptosis of canine gallbladder epithelial cells in monolayer culture.

    PubMed

    Seo, Dong Wan; Choi, Ho-Soon; Lee, Sum P; Kuver, Rahul

    2004-12-01

    Oxysterols have been detected in various mammalian organs and blood. Biliary epithelium is exposed to high concentrations of cholesterol, and we have identified three keto-oxysterols (cholest-4-en-3-one, cholesta-4,6-dien-3-one, cholesta-3,5-dien-7-one) in human bile and gallstones. Because the effects of oxysterols on biliary physiology are not well defined, we investigated their biological effects on dog gallbladder epithelial cells. Enriched medium (culture medium containing taurocholate and lecithin and cholesterol +/- various oxysterols) was applied to confluent monolayers of dog gallbladder epithelial cells in culture. Cytotoxicity and apoptosis were studied by morphological analysis and flow cytometry. Oxysterols in the mitochondrial fraction were identified by gas chromatography/mass spectrometry, whereas release of cytochrome c from mitochondria was assayed by spectrophotometry and Western blot analysis. Compared with cells treated with culture medium or with enriched medium containing cholesterol, oxysterol-treated cells showed significantly increased apoptosis (P < 0.05). Exogenously applied oxysterols were recovered from the mitochondrial fraction. Cytochrome c release from mitochondria was increased significantly by cholest-4-en-3-one, cholesta-4,6-dien-3-one, and 5beta-cholestan-3-one (all P < 0.05). Thus oxysterols recovered from human bile and gallstones induce apoptosis of biliary epithelium via a mitochondrial-dependent pathway and may play a role in the pathogenesis of chronic inflammation and carcinogenesis in the gallbladder.

  1. Identification of Regulatory Factors for Mesenchymal Stem Cell-Derived Salivary Epithelial Cells in a Co-Culture System

    PubMed Central

    Park, Yun-Jong; Koh, Jin; Gauna, Adrienne E.; Chen, Sixue; Cha, Seunghee

    2014-01-01

    Patients with Sjögren’s syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia. PMID:25402494

  2. Azithromycin: assessment of intrinsic cytotoxic effects on corneal epithelial cell cultures

    PubMed Central

    Mencucci, Rita; Pellegrini-Giampietro, Domenico E; Paladini, Iacopo; Favuzza, Eleonora; Menchini, Ugo; Scartabelli, Tania

    2013-01-01

    Purpose To compare the cytotoxic effects of preservative-free azithromycin on corneal epithelial cells in vivo with those of preservative-free netilmicin and levofloxacin, and the preservative benzalkonium chloride (BAK). Methods Rabbit corneal epithelial cells in vitro were incubated for 15 minutes or 6 hours with commercially available ophthalmic preservative-free netilmicin 0.3%, levofloxacin 0.3%, or azithromycin 1.5% preparations or different concentrations of unpreserved azithromycin and different concentrations of BAK. Qualitative analysis was undertaken using phase-contrast optics to examine the morphological aspects of cell cultures and quantitative analysis was undertaken by measuring the release of the cytoplasmic enzyme lactate dehydrogenase into the medium immediately and 24 hours after exposure to drugs. Finally, we observed the wound-healing rate of mechanically injured corneal epithelial cells exposed to each antibiotic ophthalmic preparation for 48 hours. Results Our results show that both the commercially available unpreserved mono-dose preparation of azithromycin and ophthalmic preparations of azithromycin up to a concentration of 1.5% were virtually devoid of harmful effects under our experimental conditions. This was not significantly different from the results obtained for the other antibiotic preparations (P > 0.05) tested, but was unlike the results obtained for BAK. Azithromycin 1.5% also showed good recovery properties after a mechanical wound test. Conclusion Under our experimental conditions, unpreserved azithromycin 1.5% showed a much lower toxicity than BAK and did not interfere with the wound-healing process. PMID:23737659

  3. Contact with cultured epithelial cells stimulates secretion of Salmonella typhimurium invasion protein InvJ.

    PubMed Central

    Zierler, M K; Galán, J E

    1995-01-01

    Contact of Salmonella typhimurium with cultured epithelial cells results in the assembly of surface appendages termed invasomes which are presumably required for the internalization of these organisms into host cells. The assembly of these structures requires the function of a dedicated protein secretion system encoded in the inv locus. We show in this report that contact of wild-type S. typhimurium with cultured Henle-407 cells stimulated the secretion of InvJ, a recently identified target of the inv-encoded type III protein secretion system. Stimulation of InvJ secretion also occurred upon bacterial contact with bovine calf serum-coated culture dishes but did not occur upon S. typhimurium contact with glutaraldehyde-fixed Henle-407 cells. The stimulation of InvJ secretion did not require de novo protein synthesis. Invasion-defective invC and invG mutants of S. typhimurium failed to secrete InvJ upon contact with live Henle-407 cells. In contrast, contact-dependent secretion of InvJ in S. typhimurium invE mutants occurred at levels equivalent to those of the wild type. These results indicate that the presence of Henle-407 cells and/or serum is capable of activating the type III secretion system encoded in the inv locus, further supporting the notion that Salmonella entry into cultured cells is the result of a biochemical cross-talk between the bacteria and the host cells. PMID:7558314

  4. Adenine nucleotides stimulate migration in wounded cultures of kidney epithelial cells.

    PubMed Central

    Kartha, S; Toback, F G

    1992-01-01

    Adenine nucleotides speed structural and functional recovery when administered after experimental renal injury in the rat and stimulate proliferation of kidney epithelial cells. As cell migration is a component of renal regeneration after acute tubular necrosis, we have used an in vitro model of wound healing to study this process. High density, quiescent monkey kidney epithelial cultures were wounded by mechanically scraping away defined regions of the monolayer to simulate the effect of cell loss after tubular necrosis and the number of cells that migrated into the denuded area was counted. Migration was independent of cell proliferation. Provision of adenosine, adenine nucleotides, or cyclic AMP increased the number of migrating cells and accelerated repair of the wound. Other purine and pyrimidine nucleotides were not effective. Arginine-glycine-aspartic acid-serine peptide, which blocks the binding of extracellular fibronectin to its cell surface receptor, completely inhibited migration in the presence or absence of ADP. Very low concentrations of epidermal growth factor (K0.5 approximately 0.3 ng/ml) stimulated migration, whereas transforming growth factor-beta 2 was inhibitory (Ki approximately 0.2 ng/ml). Thus, adenosine and/or adenine nucleotides released from injured or dying renal cells, or administered exogenously, may stimulate surviving cells in the wounded nephron to migrate along the basement membrane, thereby rapidly restoring tubular structure and function. Images PMID:1634617

  5. Isolation and Culture of Bovine Oviductal Epithelial Cells for Use in the Anatomy and Physiology Laboratory and Undergraduate Research

    ERIC Educational Resources Information Center

    Way, Amy L.

    2006-01-01

    This article presents methods for the isolation and culture of epithelial cells from the bovine oviduct for use in both research and the teaching laboratory and provides examples of ways that an oviductal cell culture can be incorporated into an undergraduate research program. Cow reproductive tracts are readily available from area butchers, and…

  6. Efficient and simple approach to in vitro culture of primary epithelial cancer cells

    PubMed Central

    Janik, Karolina; Popeda, Marta; Peciak, Joanna; Rosiak, Kamila; Smolarz, Maciej; Treda, Cezary; Rieske, Piotr; Stoczynska-Fidelus, Ewelina; Ksiazkiewicz, Magdalena

    2016-01-01

    Primary cancer cells constitute a favourable testing platform for in vitro research in oncology field as they reflect tumour state more accurately than the most commonly employed stable cell lines. Unfortunately, due to limited availability of material and difficulties with protocols validation, primary models are rarely implemented into laboratory practice. We have compared protocols for primary cultures, differing in media components and plate coatings. In terms of culture establishment, application of Geltrex® coating demonstrated equal efficiency to feeder layer (83% compared with 72% successfully established breast and 80% compared with 80% prostate tumour specimens), yet it was substantially less complicated and easier to validate. Both Geltrex® coating and tissue-specific primary cell medium were permanently required to successfully maintain primary epithelial prostate cancer cells (PEPCs) in culture. In case of primary epithelial breast cancer cells (PEBCs), collagen I coating enabled to obtain comparable number of passages to Geltrex® coating (P=0.438). Commercial primary cell media demonstrated lower efficiency than tissue-specific ones (PEPCs–5 compared with 8 and PEBCs–6 compared with 9 passages). Interestingly, both analysed tumour types were unsusceptible to induction of culture lifespan extension when transduced with SV40LT, BMI-1 or hEST2 genes, commonly applied as potential immortalizing agents. In conclusion, the approach based on extracellular matrix reconstitution and tissue-specific primary cell media is easy to validate and provides in vitro expansion sufficient for analytical purposes (approximately 8 passages). Therefore, it may facilitate implementation of hardly available experimental models for a variety of analyses. PMID:27803125

  7. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts.

    PubMed

    Aslanova, Afag; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Yamamoto, Masakazu

    2015-05-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell-cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  8. PRIMARY CULTURE OF CHOROIDAL EPITHELIAL CELLS: CHARACTERIZATION OF AN IN VITRO MODEL OF BLOOD-CSF BARRIER

    PubMed Central

    ZHENG, WEI; ZHAO, QIUQU; GRAZIANO, JOSEPH H.

    2016-01-01

    Summary A primary rat choroidal epithelial cell culture system was developed to investigate mechanisms of heavy metal toxicity on the blood-cerebrospinal fluid (CSF) barrier. Epithelial cells were dissociated from choroidal tissue by pronase digestion and cultured in standard DMEM culture media supplemented with 10% fetal bovine serum and 10 ng epithelial growth factor per ml. The procedure yielded 2–5 × 104 cells from pooled plexuses of three to four rats, and a viability of 77–85%. The cultures displayed a dominant polygonal type of epithelial cells, with a population doubling time of 2–3 d. The cultures were of distinct choroidal epithelial origins. For example, immunocytochemical studies using monospecific rabbit anti-rat TTR polyclonal antibody revealed a strong positive stain of transthyretin (TTR), a thyroxine transport protein exclusively produced by the choroidal epithelia. Also, reverse-transcriptase polymerase chain reaction (PCR) confirmed the presence of specific TTR mRNA in the cultures. The cultures were further adapted to grow on a freely permeable membrane sandwiched between two culture chambers. The formation of an impermeable confluent monolayer occurred within 5 d after seeding and was verified by the presence of a steady electrical resistance across the membrane (80 ± 10 ohm per cm2). The epithelial barriers appeared to actively transport [125I]-thyroxine from the basal to apical chamber. These results suggest that this primary cell culture system possesses typical choroidal epithelial characteristics and appears to be a suitable model for in vitro mechanistic investigations of blood–CSF barrier. PMID:9542634

  9. Isolation, culture and phenotypic characterization of human sweat gland epithelial cells.

    PubMed

    Gao, Yunhe; Li, Meiying; Zhang, Xueyan; Bai, Tingting; Chi, Guanfan; Liu, Jin Yu; Li, Yulin

    2014-10-01

    Sweat gland epithelial cells (SGECs) have been identified as essential for the regeneration of sweat glands and for the construction of skin substitutes containing skin appendages. Consequently, the isolation, culture and phenotypic characterization of SGECs are of paramount importance. In the present study study, human sweat glands were isolated by pipetting under a phase contrast microscope following digestion with collagenase type I. Subsequently, a microscopic organ culture technique was used for the primary culture of human SGECs, and the culture conditions were modified in order to achieve optimal cell growth status. Primary SGECs were identified based on their expression of markers specific for sweat glands, including carcinoembryonic antigen (CEA), CK7, CK8, CK14, CK15, CK18 and CK19. We explored the possible presence of stem cells in human sweat glands by detecting their expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5). Primary SGECs achieved a good growth state when cultured under serum-free conditions. After one passage, the cells cultured in keratinocyte serum-free medium with 1% fetal bovine serum (FBS) still showed a prominent proliferative activity. Phenotypic analysis by immunofluorescence microscopy, reverse transcription-polymerase chain reaction (RT-PCR), and western blot analysis demonstrated the expression of sweat gland-specific markers, including CEA, CK7, CK8, CK14, CK15, CK18 and CK19. In addition, RT-PCR and immunochemistry detected the expression of LGR5. In comparison with traditional serum-containing conditions, serum-free culture provides the preferred culture conditions for human SGECs. LGR5 is a novel marker that identifies human sweat gland-derived stem cells.

  10. Increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis.

    PubMed Central

    Cheng, P W; Boat, T F; Cranfill, K; Yankaskas, J R; Boucher, R C

    1989-01-01

    Cystic fibrosis (CF) respiratory epithelia exhibit abnormal anion transport that may be linked to abnormal lung defense. In these studies, we investigated whether primary cultures of CF respiratory epithelial cells regulate abnormally the sulfate content of high molecular weight glycoconjugates (HMG) participating in airways' mucosal defense. HMG, including glycosaminoglycans and mucin-type glycoproteins released spontaneously into medium and HMG released from cell surfaces by trypsin, were metabolically labeled with 35SO4- and [6-3H]-glucosamine (GlcN) or 35SO4- and [3H]serine. All three classes of HMG from CF cells exhibited 35S/3H labeling ratios 1.5-4-fold greater than HMG from normal or disease control cells. Differences for labeling ratios of HMG from CF cells were shown to be the consequence of increased 35SO4- incorporation rather than decreased peptide synthesis and release or HMG glycosylation. The buoyant density of CF mucin-type HMG also was increased, consistent with increased sulfation. These observations suggest that oversulfation of a spectrum of HMG is a genetically determined characteristic of CF epithelial cells and may play an important pathophysiological role by altering the properties of mucous secretions and/or the interactions between selected bacteria and HMG at the airways' surface. Images PMID:2738159

  11. Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells.

    PubMed

    Sundqvist, K; Liu, Y; Nair, J; Bartsch, H; Arvidson, K; Grafström, R C

    1989-10-01

    Because betel quid chewing has been linked to the development of oral cancer, pathobiological effects of an aqueous areca nut extract, four areca nut alkaloids (arecoline, guvacoline, guvacine, and arecaidine), and four nitrosated derivatives [N-nitrosoguvacoline, N-nitrosoguvacine, 3-(N-nitrosomethylamino)propionaldehyde and 3-(N-nitrosomethylamino)propionitrile] have been investigated using cultured human buccal epithelial cells. Areca nut extract in a dose-dependent manner decreases cell survival, vital dye accumulation, and membrane integrity, and it causes formation of both DNA single strand breaks and DNA protein cross-links. Depletion of cellular free low-molecular-weight thiols also occurs, albeit at quite toxic concentrations. Comparisons of the areca nut-related N-nitroso compounds and their precursor alkaloids, at concentrations up to 5 mM, indicate that 3-(N-nitrosomethylamino)propionaldehyde is the most potent on a molar basis to decrease both survival and thiol content and to cause significant formation of DNA single strand breaks. Arecoline, guvacoline, or N-nitrosoguvacoline decreases survival and cellular thiols, whereas arecaidine, guvacine, N-nitrosoguvacine, and 3-(N-nitrosomethylamino)propionitrile have only minor effects on these variables. Taken together, the present studies indicate that aqueous extract and, in particular, one N-nitroso compound related to areca nut, i.e., 3-(N-nitrosomethylamino)propionaldehyde, are highly cytotoxic and genotoxic to cultured human buccal epithelial cells, of potential importance in the induction of tumors in betel quid chewers.

  12. Establishment of mammary gland model in vitro: culture and evaluation of a yak mammary epithelial cell line.

    PubMed

    Fu, Mei; Chen, Yabing; Xiong, Xianrong; Lan, Daoliang; Li, Jian

    2014-01-01

    This study aimed to establish yak mammary epithelial cells (YMECs) for an in vitro model of yak mammary gland biology. The primary culture of YMECs was obtained from mammary gland tissues of lactating yak and then characterized using immunocytochemistry, RT-PCR, and western blot analysis. Whether foreign genes could be transfected into the YMECs were examined by transfecting the EGFP gene into the cells. Finally, the effect of Staphylococcus aureus infection on YMECs was determined. The established YMECs retained the mammary epithelial cell characteristics. A spontaneously immortalized yak mammary epithelial cell line was established and could be continuously subcultured for more than 60 passages without senescence. The EGFP gene was successfully transferred into the YMECs, and the transfected cells could be maintained for a long duration in the culture by continuous subculturing. The cells expressed more antimicrobial peptides upon S.aureus invasion. Therefore, the established cell line could be considered a model system to understand yak mammary gland biology.

  13. Concise review: transplantation of cultured oral mucosal epithelial cells for treating limbal stem cell deficiency-current status and future perspectives.

    PubMed

    Utheim, Tor Paaske

    2015-06-01

    A number of diseases and external factors can deplete limbal stem cells, causing pain and visual loss. Ten years have passed since the first transplantation of cultured oral mucosal epithelial cells in humans, representing the first autologous cell-based therapy for severe bilateral limbal stem cell deficiency. Its steady increase in popularity since then can be attributed to the accumulating evidence of its efficacy in reverting limbal stem cell deficiency. In this review, the focus is on clinical, and to a lesser degree laboratory, features of cultured oral mucosal epithelial transplants over the past 10 years. Comparisons with other available technologies are made. Avenues for research to stimulate further improvements in clinical results and allow worldwide distribution of limbal stem cell therapy based on oral mucosal cells are discussed. These include storage and transportation of cultured oral mucosal epithelial sheets and in vivo culture of oral mucosal epithelial cells.

  14. Isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications.

    PubMed

    Murphy, Sean V; Kidyoor, Amritha; Reid, Tanya; Atala, Anthony; Wallace, Euan M; Lim, Rebecca

    2014-12-21

    Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success. hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

  15. Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies.

    PubMed

    Moura, Marcos de Assis; Amendoeira, Maria Regina Reis; Barbosa, Helene Santos

    2009-09-01

    The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

  16. Human airway epithelial cell culture to identify new respiratory viruses: coronavirus NL63 as a model.

    PubMed

    S Banach, Bridget; Orenstein, Jan M; Fox, Linda M; Randell, Scott H; Rowley, Anne H; Baker, Susan C

    2009-03-01

    Propagation of new human respiratory virus pathogens in established cell lines is hampered by a lack of predictability regarding cell line permissivity and by availability of suitable antibody reagents to detect infection in cell lines that do not exhibit significant cytopathic effect. Recently, molecular methods have been used to amplify and identify novel nucleic acid sequences directly from clinical samples, but these methods may be hampered by the quantity of virus present in respiratory secretions at different time points following the onset of infection. Human airway epithelial (HAE) cultures, which effectively mimic the human bronchial environment, allow for cultivation of a wide variety of human respiratory viral pathogens. The goal of the experiments described here was to determine if propagation and identification of a human respiratory virus may be achieved through inoculation of HAE cultures followed by whole transcriptome amplification (WTA) and sequence analysis. To establish proof-of-principle human coronavirus NL63 (HCoV-NL63) was evaluated, and the first visualization of HCoV-NL63 virus by transmission electron microscopy (TEM) is reported. Initial propagation of human respiratory secretions onto HAE cultures followed by TEM and WTA of culture supernatant may be a useful approach for visualization and detection of new human respiratory pathogens that have eluded identification by traditional approaches.

  17. Epithelial cell culture models for the prevention and therapy of clinical breast cancer (Review)

    PubMed Central

    2012-01-01

    Clinical breast cancer progresses via a multi-step carcinogenic process wherein genetic, molecular, endocrine and dietary factors play significant roles in the pathogenesis, prevention and therapy of the disease. Preclinical cell culture models, expressing clinically relevant genetic and endocrine defects and exhibiting quantifiable cancer risk, may provide facile, clinically translatable approaches to identify molecular targets and susceptible mechanistic pathways for the efficacy of novel interventional approaches. This review summarizes laboratory investigations focused on i) developing murine and human mammary tissue-derived cell culture models; ii) optimizing mechanism-based quantitative endpoint biomarker assays specific for carcinogenic risk and preventive/therapeutic efficacy; and iii) providing quantifiable proof-of-principle evidence for validation of the present cell culture approaches, capable of prioritizing efficacious lead compounds for subsequent in vivo animal studies and clinical trials for the prevention/therapy of breast cancer. Epithelial cell culture models are developed and characterized where the carcinogenic process is initiated by the targeted expression of clinically relevant oncogenes. The cell culture systems from mouse mammary tissue are in vitro approaches that complement the Ras and Myc transgenic mouse models. The human mammary tissue-derived systems are in vitro models for chemoendocrine, therapy-resistant, clinical, pre-invasive ER-/PR-/HER-2+ comedo ductal carcinoma in situ, ER+/PR+ chemoendocrine therapy-responsive breast cancer and ER-/PR-/HER-2- triple-negative chemoendocrine, therapy-resistant breast cancer. The oncogene-initiated phenotypes exhibit loss of homeostatic growth control, downregulation of cell apoptosis and gain of carcinogenic risk in vitro, as well as transplantable tumor development in vivo. Numerous mechanistically distinct, synthetic pharmacological agents, as well as naturally occurring dietary compounds

  18. Cationorm shows good tolerability on human HCE-2 corneal epithelial cell cultures.

    PubMed

    Kinnunen, Kati; Kauppinen, Anu; Piippo, Niina; Koistinen, Arto; Toropainen, Elisa; Kaarniranta, Kai

    2014-03-01

    Preservatives have been for a long time known to cause detrimental effects on ocular surface. Cationorm, a preservative-free compound with electrostatic properties is a novel way to solve the problems encountered with traditional benzalkonium chloride (BAK)-containing eye drops. The aim of this study was to evaluate tolerability of the preservative-free cationic emulsion Cationorm in vitro on corneal epithelial cells. The human corneal epithelial cell (HCE-2) culture line was used to study cellular morphology, cytotoxicity and inflammatory responses after Cationorm diluted 1/10 exposure for 5, 15 and 30 min. Exposures to Systane diluted 1/10 with polyquaternium-1/polidronium chloride 0.001% as preservative, BAK 0.001% or C16 (0.0002%) and normal cell culture medium served as positive and negative references. Cell viability was determined by measuring the release of lactate dehydrogenase (LDH) and mitochondrial dehydrogenase activity was evaluated using 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The possible induction of apoptosis was analyzed by measuring the activity of caspase-3, and Cell Counting Kit-8 (CCK-8) was used to evaluate the number of viable cells after the exposure to test compounds. Furthermore, the tendency of the test compounds to produce inflammatory reaction was determined by analyzing the production of proinflammatory cytokines IL-6 and IL-8, and DNA binding of the p65 subunit of transcription factor NF-κB was measured from cell lysates. HCE-2 cells showed no morphological changes after the exposure to Cationorm, but in cells exposed to BAK, clear cytoplasm vacuolization and loose cell-cell contacts were observed in transmission (TEM) or scanning (SEM) electron microscopic analyses. Cell viability, as measured with the release of LDH, indicated a time dependent increase in LDH expression after exposure to all test compounds but especially with BAK. Moreover, Cationorm and BAK time-dependently decreased the

  19. Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

    PubMed

    Jansen, J; De Napoli, I E; Fedecostante, M; Schophuizen, C M S; Chevtchik, N V; Wilmer, M J; van Asbeck, A H; Croes, H J; Pertijs, J C; Wetzels, J F M; Hilbrands, L B; van den Heuvel, L P; Hoenderop, J G; Stamatialis, D; Masereeuw, R

    2015-11-16

    The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

  20. Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations

    PubMed Central

    Jansen, J.; De Napoli, I. E; Fedecostante, M.; Schophuizen, C. M. S.; Chevtchik, N. V.; Wilmer, M. J.; van Asbeck, A. H.; Croes, H. J.; Pertijs, J. C.; Wetzels, J. F. M.; Hilbrands, L. B.; van den Heuvel, L. P.; Hoenderop, J. G.; Stamatialis, D.; Masereeuw, R.

    2015-01-01

    The bioartificial kidney (BAK) aims at improving dialysis by developing ‘living membranes’ for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as a fluorescent substrate. Initial ASP+ uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a ‘living membrane’ of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering. PMID:26567716

  1. Epithelialization and stromalization of porcine follicular granulosa cells during real-time proliferation - a primary cell culture approach.

    PubMed

    Ciesiółka, S; Bryja, A; Budna, J; Kranc, W; Chachuła, A; Bukowska, D; Piotrowska, H; Porowski, L; Antosik, P; Bruska, M; Brüssow, K P; Nowicki, M; Zabel, M; Kempisty, B

    2016-01-01

    The process of oocyte growth and development takes place during long stages of folliculogenesis and oogenesis. This is accompanied by biochemical and morphological changes, occurring from the preantral to antral stages during ovarian follicle differentiation. It is well known that the process of follicle growth is associated with morphological modifications of theca (TCs) and granulosa cells (GCs). However, the relationship between proliferation and/or differentiation of porcine GCs during long-term in vitro culture requires further investigation. Moreover, the expression of cytokeratins and vimentin in porcine GCs, in relation to real-time cell proliferation, has yet to be explored. Utilizing confocal microscopy, we analyzed cytokeratin 18 (CK18), cytokeratin 8 + 18 + 19 (panCK), and vimentin (Vim) expression, as well as their protein distribution, within GCs isolated from slaughtered ovarian follicles. The cells were cultured for 168 h with protein expression and cell proliferation index analyzed at 24-h intervals. We found the highest expression of CK18, panCK, and Vim occurred at 120 h of in vitro culture (IVC) as compared with other experimental time intervals. All of the investigated proteins displayed cytoplasmic distribution. Analysis of real-time cell proliferation revealed an increased cell index after the first 24 h of IVC. Additionally, during each period between 24-168 h of IVC, a significant difference in the proliferation profile, expressed as the cell index, was also observed. We concluded that higher expression of vimentin at 120 h of in vitro proliferation might explain the culmination of the stromalization process associated with growth and domination of stromal cells in GC culture. Cytokeratin expression within GC cytoplasm confirms the presence of epithelial cells as well as epithelial-related GC development during IVC. Moreover, expression of both cytokeratins and vimentin during short-term culture suggests that the process of GC proliferation

  2. Ozone enhances the uptake of mineral particles by tracheobronchial epithelial cells in organ culture.

    PubMed

    Churg, A; Brauer, M; Keeling, B

    1996-04-01

    We have previously shown that the basal uptake of mineral particles by tracheobronchial epithelial cells in organ culture is mediated in part by active oxygen species (AOS) and can be greatly augmented by exposure to cigarette smoke, a concentrated source of AOS, and other radicals. We hypothesized that ozone, another generator of AOS in tissues, might have the same effect. To test this hypothesis, tracheal explants were exposed to room air (control) or ozone in varying concentrations from 0.01 to 1.0 ppm for 10 min, and subsequently to a suspension of either amosite asbestos or titanium dioxide (rutile) for 1 h. Explants were then transferred to an air/CO2 incubator for 1 wk to allow particle uptake to occur, and uptake was determined by morphometry. We found that ozone exposure increased the uptake of both asbestos and titanium dioxide in a dose-response fashion; this effect appeared at lower exposure levels and was more marked with titanium dioxide than with amosite. The ozone effect could be prevented by addition of catalase but not superoxide dismutase to the particle suspension, or by preincubation of the particles with deferoxamine. These observations indicate that ozone can directly increase uptake of mineral particles by tracheobronchial epithelial cells; this effect occurs with brief exposures at very low ozone levels and appears to be mediated by hydrogen peroxide and possibly by hydroxyl radical. These findings support the general hypothesis that AOS are important mediators of epithelial particle uptake in many different settings. Enhanced uptake may be one of the mechanisms by which ozone impairs particle clearance from the lung and may play a role in the increased morbidity seen in populations with exposure to high levels of both ozone and atmospheric particulates.

  3. Proteins with epitopes of the acetylcholine receptor in epithelial cell cultures of thymomas in myasthenia gravis.

    PubMed Central

    Marx, A.; Kirchner, T.; Hoppe, F.; O'Connor, R.; Schalke, B.; Tzartos, S.; Müller-Hermelink, H. K.

    1989-01-01

    Thymomas from 12 patients with myasthenia gravis (MG) were investigated for the presence of epitopes of the alpha-subunit of the nicotinic acetylcholine receptor (AchR) using monoclonal antibodies (MAb) reacting against the AchR. In all but two of the tumors epitopes corresponding to antigenic determinants located on the cytoplasmic side of the AchR were identified. From eight thymomas cell lines were established that have been kept in culture for up to 6 months. The cultured cells expressed the same AchR-epitopes as did the primary tumors. During early passages the percentage of epithelial cells positive for the AchR epitopes approximately mirrored the percentage of positive cells in the original tumors. With passaging the relative number of positive cells usually declined but in some cultures an increase was observed. Three cell lines that showed extensive staining with an MAb against the AchR were radiolabeled to characterize the antigen. From protein extracts of these three cell lines proteins of 45 kd and 156 kd molecular weight (MW) were precipitated. These proteins are different from other proteins described in the context of both thymomas and MG. The negative reactivity with MAb against other epitopes of the alpha-subunit, especially against the main immunogenic region (MIR), speaks in favor of membrane-associated proteins of only limited crossreactivity to the AchR. A previous study found an almost exclusive occurrence of these AchR-epitopes in thymomas associated with MG, but not in other thymomas of similar histologic type. The expression of the proteins described here could therefore play a role in the triggering of the autoimmune process against the AchR of the motor, endplate in MG patients. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2468286

  4. Uptake and esterification of vitamin A by RCS rat retinal pigment epithelial cells in primary culture.

    PubMed

    Cia, David; Bonhomme, Brigitte; Azaïs-Braesco, Véronique; Cluzel, Jacques; Doly, Michel

    2004-02-01

    We investigated the capacity of Royal College of Surgeons (RCS) rat retinal pigment epithelial (RPE) cells to take up all-trans-retinol (ROL) (vitamin A) and to metabolize it into retinyl esters (RE). Cultures of RPE cells were established from RCS and control newborn rats. All-trans-ROL was delivered to the apical surface of the RPE monolayer. Retinoids were analyzed by high-performance liquid chromatography. The cellular retinol-binding protein type I (CRBP-I) was assessed by Western blotting. Before supplementation with ROL, RE were lower in RCS rats. After ROL supplementation, esters increased and reached values that were similar in the two strains, but the increase, expressed relative to the initial value, was higher in RCS rats. The uptake of ROL and the level of CRBP-I were greater in RCS rats. Our results provide evidence of a functional retinol esterifying enzyme in cultured RCS RPE cells and suggest that CRBP-I could play a role in the uptake and esterification of ROL in the RPE cells.

  5. Mucin biosynthesis and secretion in tracheal epithelial cells in primary culture.

    PubMed Central

    Svitacheva, N; Davies, J R

    2001-01-01

    Density-gradient centrifugation of bovine tracheal epithelial cell extracts revealed a 'high-density' (1.48 g/ml) sialic-acid-rich population as well as a 'low-density' (1.42 g/ml) one that reacted more strongly with a periodate-Schiff (PAS) assay. The sialic-acid-rich mucins were oligomeric molecules containing disulphide- bond-linked subunits and large glycosylated domains, whereas the PAS-reactive component seemed to be smaller and 'monomeric'. Only the 'high-density' population was secreted from cells cultured for 5 days on plastic or a collagen type 1, Matrigel or Vitrogen substrate. Release was less from cells grown on plastic than from those on a substrate and the amount was unaffected by increasing the thickness of the collagen layer. For cells grown on collagen, the amount of the sialic-acid-rich mucin increased over 10 days, whereas the PAS-reactive component was largely absent after 24 h, which was consistent with an initial release of stored PAS-reactive molecules and synthesis of the sialic-acid-rich mucins de novo. Both [(3)H]proline and [(35)S]sulphate were poorly incorporated into mucins detected with the chemical assays but molecules with a higher buoyant density than that of either of the previously identified species were labelled with [(35)S]sulphate. The [(35)S]sulphate-labelled material yielded large trypsin-resistant fragments and contained O-linked glycans but was not affected by digestion with chondroitin ABC lyase or heparan sulphate lyase, suggesting that it is a mucin rather than a proteoglycan. [(35)S]Sulphate is thus a poor marker for the major oligomeric mucins produced by bovine tracheal epithelial cells but the radiolabel is incorporated into a heavily labelled mucin-like component. PMID:11115395

  6. Neuropeptides Exert Direct Effects on Rat Thymic Epithelial Cells in Culture

    PubMed Central

    Head, Gail M.; Mentlein, R.; Patay, Birte Von; Downing, J. E.G.

    1998-01-01

    To determine if major thymic neuropeptides and neurotransmitters can directly influence the functional activity of cultured rat thymic epithelium, neuropeptides and neurotransmitters were applied, and intercellular communication, proliferation, and thymulin secretion assessed. After injections of a mixture of lucifer yellow dextran (too large to pass gap junctions) and cascade blue (which does) into single cells, some neuropeptides decrease dye coupling: 0.1 mM GABA (P < 0.0001), 100 nM NPY (P < 0.0001), 100 nM VIP (P < 0.001), 100 nM CGRP (P < 0.001), 100 nM SP (P < 0.01), and 0.1 mM histamine (P < 0.01), whereas 0.1 mM 5-HT, mM acetylcholine, and 1 μM isoproterenol (β-adrenergic agonist) had no effect. Proliferation (incorporation of tritiated thymidine) was increased by CGRP (P = 0.004) and histamine (P < 0.02), but decreased by isoproterenol (P = 0.002), 5-HT (P = 0.003), and acetylcholine (P < 0.05). The percentage of multinucleate cells was decreased after isoproterenol (2.5%), and increased after 5-HT (21.3%), GABA (15%), and histamine (15.1%). Compared to controls, thymulin in the supernatant was decreased after challenge with acetylcholine (52%), isoproterenol (71%), 5-HT (73%), and histamine (84%). This study demonstrates direct effects of neuropeptides and neurotransmitters on functional aspects of cultured thymic epithelial cells. PMID:9716910

  7. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

    SciTech Connect

    Aslanova, Afag; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Yamamoto, Masakazu

    2015-05-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  8. Establishment of a novel lingual organoid culture system: generation of organoids having mature keratinized epithelium from adult epithelial stem cells.

    PubMed

    Hisha, Hiroko; Tanaka, Toshihiro; Kanno, Shohei; Tokuyama, Yoko; Komai, Yoshihiro; Ohe, Shuichi; Yanai, Hirotsugu; Omachi, Taichi; Ueno, Hiroo

    2013-11-15

    Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

  9. Establishment of a Novel Lingual Organoid Culture System: Generation of Organoids Having Mature Keratinized Epithelium from Adult Epithelial Stem Cells

    NASA Astrophysics Data System (ADS)

    Hisha, Hiroko; Tanaka, Toshihiro; Kanno, Shohei; Tokuyama, Yoko; Komai, Yoshihiro; Ohe, Shuichi; Yanai, Hirotsugu; Omachi, Taichi; Ueno, Hiroo

    2013-11-01

    Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

  10. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    PubMed

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  11. Voltage-activated currents recorded from rabbit pigmented ciliary body epithelial cells in culture.

    PubMed Central

    Fain, G L; Farahbakhsh, N A

    1989-01-01

    1. The whole-cell recording mode of the patch-clamp technique was used to investigate the presence of voltage-activated currents in the isolated pigmented cells from the rabbit ciliary body epithelium grown in culture. 2. In Ringer solution with composition similar to that of the rabbit aqueous humour, depolarizing voltage steps activated a transient inward current and a delayed outward current, while hyperpolarization elicited an inwardly rectified current. 3. The depolarization-activated inward current was mainly carried by Na+ and was blocked by submicromolar concentrations of tetrodotoxin. This current in many cells was sufficiently large to produce a regenerative Na+ spike. 4. The depolarization-activated outward current was carried by K+ and blocked by external TEA and Ba2+. Its activation appeared to be Ca2(+)-independent. 5. The hyperpolarization-activated inward current was almost exclusively carried by K+ and was blocked by Ba2+ and Cs+. For large hyperpolarizations below -120 mV, this current exhibited a biphasic activation with a fast transient peak followed by a slower sag, that appeared to be due to K+ depletion. 6. The voltage-dependent K+ conductances probably act to stabilize the cell membrane resting potential and may also play a role in ion transport. The function of the Na(+)-dependent inward current is unclear, but it may permit the electrically coupled epithelial cells of the ciliary body to conduct propagated action potentials. Images Fig. 2 PMID:2621623

  12. EFFECTS OF ATRAZINE AND AN ATRAZINE METABOLITE MIXTURE ON DIFFERENTIATED MAMMARY EPITHELIAL CELL MILK PROTEIN PRODUCTION IN CULTURE

    EPA Science Inventory

    Effects of Atrazine and an Atrazine Metabolite Mixture on Differentiated Mammary Epithelial Cell Milk Protein Production in Culture

    E.P. Hines, R. Barbee, M. Blanton, M.S. Pooler, and S.E. Fenton. US EPA, ORD/NHEERL, RTD, RTP, NC, 27711, USA.

    Previous studies have ...

  13. Interaction of platelet-activating factor with cultured guinea pig tracheal epithelial cells.

    PubMed Central

    Churchill, L; Chilton, F H; Proud, D

    1991-01-01

    The present study has examined the interaction of platelet-activating factor (PAF) with cultured guinea pig tracheal epithelial cells (GTE). PAF stimulated GTE to release endogenous arachidonic acid and metabolize it to prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha). Prostanoid production by GTE in response to PAF was dose-dependent (0.1-100 nM) and was maximal within 5 min. PGE2 and PGF2 alpha levels increased by 3.3 +/- 0.8 and 3.2 +/- 0.6 ng/10(6) cells respectively over basal levels in response to 100 nM-PAF. The ability of GTE to synthesize and/or catabolize PAF was also examined. GTE readily incorporated [3H]acetate into a product which migrated on t.l.c. with PAF. However, further characterization of this product suggested that label had not been incorporated into PAF, but rather that it was incorporated into another lipid product with chromatographic characteristics similar to those of PAF. In contrast, GTE readily metabolized PAF to inactive products. When [3H]PAF was incubated with GTE, 50% of the total [3H]PAF added was catabolized in approx. 15 min. The major route of catabolism of PAF by GTE was the deacetylation-reacylation pathway, which yielded 1-O-[3H]alkyl-2-acyl-sn-glycerophosphocholine. Determination of the nature of the long-chain acyl group incorporated into the sn-2 position of the newly synthesized products revealed that oleic and linoleic acids were the major fatty acids present. Taken together, these results suggest that respiratory epithelial cells respond to stimulation by PAF with enhanced production of PGE2 and PGF2 alpha, and also have the capacity to modulate inflammatory reactions in the airways by their ability to degrade this potent inflammatory mediator. Images Fig. 1. PMID:2064601

  14. Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

    SciTech Connect

    Katano, Takahito; Ootani, Akifumi; Mizoshita, Tsutomu; Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Ebi, Masahide; Mori, Yoshinori; Kataoka, Hiromi; Kamiya, Takeshi; Toda, Shuji; Joh, Takashi

    2013-03-22

    Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.

  15. Vortex or whorl formation of cultured human corneal epithelial cells induced by magnetic fields.

    PubMed

    Dua, H S; Singh, A; Gomes, J A; Laibson, P R; Donoso, L A; Tyagi, S

    1996-01-01

    The terms 'vortex keratopathy' and 'hurricane keratopathy' describe two similar conditions affecting the corneal surface. In the former, a vortex or whorl pattern is seen on the corneal surface and is due to the deposition of substances such as pigment, iron or drugs in the epithelial cells. In the latter, a similar pattern is presented by migrating epithelial cells but, unlike the former, the pattern is rendered more visible by fluorescein staining. Both represent the migratory pattern of normal epithelial cells which is otherwise not visible due to the slow rate of epithelial turnover and migration. The whorl pattern has a clockwise predisposition in the majority of cases and is hypothesised to be due to the influence of ocular electro-magnetic fields on the migrating epithelial cells. In this study we tested in vitro the effect of static magnetic fields on corneal epithelial cells. We were able to reproduce dramatic vortex or whorl patterns in response to magnetic fields, but without preferential migration towards the North or South Pole.

  16. Cytoprotective role of taurine in a renal epithelial cell culture model.

    PubMed

    Eppler, Bärbel; Dawson, Ralph

    2002-03-15

    Taurine (TAU) is a sulfur-containing amino acid that has been shown to decrease during aging and is believed to be important for cytoprotection. A decrease in TAU could exacerbate the accumulation of free radical-induced damage that may lead to cell death during the aging process. We have shown previously that TAU directly inhibits dopamine (DA) and (-)-3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) oxidation. Experiments were conducted to establish a cytoprotective role for TAU. Porcine renal epithelial cells were treated for 1 hr with iron and catecholamines (L-dopa and DA) to produce cytotoxicity by a free radical and quinone mechanism in the absence and presence of 10 or 20mM TAU. Viability assays, protein, and DNA measurements were performed after a 24hr recovery period. In some experiments, cells were extracted immediately after the insult for DA and TAU content measurements using high performance liquid chromatography with electrochemical detection. Catecholamine-induced cytotoxicity caused a 50% loss in cell viability, and 10 or 20mM TAU provided significant protection from cytotoxicity and maintained the functional integrity of the cells. Photomicrographs showed attenuation in cell loss and swelling in the presence of TAU. Pretreatment with 1mM TAU followed by exposure to iron and L-dopa in the presence of 1mM TAU caused a moderate but non-significant increase in cell survival. These data conclusively show that TAU can play a cytoprotective role in the LLC-PK(1) cell culture model.

  17. Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

    PubMed Central

    González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

    2013-01-01

    Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

  18. Monoclonal antibodies to an epithelial ovarian adenocarcinoma: distinctive reactivity with xenografts of the original tumor and a cultured cell line.

    PubMed

    Baumal, R; Law, J; Buick, R N; Kahn, H; Yeger, H; Sheldon, K; Colgan, T; Marks, A

    1986-08-01

    Four monoclonal antibodies (mAb) (8C, 10B, M2A, and M2D) were produced against the human epithelial ovarian adenocarcinoma cell line, HEY. The affinity constants of binding of the mAb to cultured HEY cells were 8 X 10(8) M-1 (M2D) and 10(9) M-1 (8C and 10B). mAb 8C reacted with a major glycoprotein of Mr 90,000 on the surface of HEY cells. The four mAb differed from previously reported mAb to epithelial ovarian adenocarcinomas on the basis of their reactivity with cultured ovarian adenocarcinoma cell lines using a cell-binding radioimmunoassay, and their staining of cryostat sections of various human normal and tumor tissues using an immunoperoxidase reaction. All four mAb reacted with s.c. tumors derived by injecting cultured HEY cells into thymectomized CBA/CJ mice. However, only two of the four mAb (8C and 10B) also reacted with s.c. tumors of the original HEY xenograft from which the cultured cell line was derived. In addition, mAb 8C and 10B reacted by immunoperoxidase staining with 2 and 4 different cases, respectively, of 11 epithelial ovarian adenocarcinomas examined. Cultured HEY cells were adapted to grow i.p. in BALB/c-nu/nu mice and the i.p. tumors retained their reactivity with the monoclonal antibodies. These tumor-bearing mice offer a useful model system for studying the potential of mAb, especially 8C and 10B, for the diagnosis and treatment of patients with peritoneal extension of epithelial ovarian adenocarcinomas.

  19. Ambroxol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells.

    PubMed

    Yamaya, Mutsuo; Nishimura, Hidekazu; Nadine, Lusamba Kalonji; Ota, Chiharu; Kubo, Hiroshi; Nagatomi, Ryoichi

    2014-04-01

    The mucolytic drug ambroxol hydrochloride reduces the production of pro-inflammatory cytokines and the frequency of exacerbation in patients with chronic obstructive pulmonary disease (COPD). However, the inhibitory effects of ambroxol on rhinovirus infection, the major cause of COPD exacerbations, have not been studied. We examined the effects of ambroxol on type 14 rhinovirus (RV14) infection, a major RV group, in primary cultures of human tracheal epithelial cells. RV14 infection increased virus titers and cytokine content in the supernatants and RV14 RNA in the cells. Ambroxol (100 nM) reduced RV14 titers and cytokine concentrations of interleukin (IL)-1β, IL-6 and IL-8 in the supernatants and RV14 RNA in the cells after RV14 infection, in addition to reducing susceptibility to RV14 infection. Ambroxol also reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for RV14, and the number of acidic endosomes from which RV14 RNA enters the cytoplasm. In addition, ambroxol reduced the activation of the transcription factor nuclear factor kappa B (NF-κB) in the nucleus. These results suggest that ambroxol inhibits RV14 infection partly by reducing ICAM-1 and acidic endosomes via the inhibition of NF-κB activation. Ambroxol may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection.

  20. Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

    NASA Technical Reports Server (NTRS)

    Craise, L. M.; Prioleau, J. C.; Stampfer, M. R.; Rhim, J. S.; Yang, TC-H (Principal Investigator)

    1992-01-01

    For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude mice. Neoplastic transformation was achieved by irradiating cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level.

  1. Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

    SciTech Connect

    Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.; Stampfer, M.R.; Rhim, J.S.

    1990-11-01

    For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude nice. Neoplastic transformation was achieved by irradiation cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level. 15 refs., 9 figs., 2 tabs.

  2. Chromosomal changes in cultured human epithelial cells transformed by low- and high-let radiation

    NASA Astrophysics Data System (ADS)

    Chui-Hsu Yang, Tracy; Craise, Laurie M.; Prioleau, John C.; Stampfer, Martha R.; Rhim, Johng S.

    1992-07-01

    For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude mice. Neoplastic transformation was achieved by irradiating cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level.

  3. Electrical properties of dog tracheal epithelial cells grown in monolayer culture.

    PubMed

    Coleman, D L; Tuet, I K; Widdicombe, J H

    1984-03-01

    Epithelial cells from dog trachea, when grown in tissue culture, formed confluent monolayers 5-6 days after plating. After 8-10 days, domes [mean diam 356 +/- (SE) 53 micron] appeared in monolayers grown in collagen-coated flasks. When grown on polycarbonate filters coated with collagen, a detectable resistance (greater than 5 omega X cm2) and transepithelial potential difference (PD) (greater than 0.1 mV) developed 6 days after plating and increased to approximately 15 omega X cm2 and 15 mV at 10 days. Serosal ouabain (10(-4) M) abolished PD and short-circuit current (Isc). Luminal ouabain had no effect. Luminal amiloride (10(-4) M) and serosal bumetanide (10(-4) M) each decreased PD and Isc. However, a combination of both of these drugs did not abolish Isc. Isoproterenol (10(-5) M), dibutyryl adenosine 3',5'-cyclic monophosphate (10(-3) M), vasoactive intestinal peptide (10(-7) M), prostaglandin (PG) E2 (10(-5) M), PGF2 alpha (10(-5) M), and bradykinin (10(-5) M) each increased PD and Isc. Thus these monolayer cultures maintain electrical properties resembling those of the original tissue. This preparation may prove useful for the study of water and ion transport by airway epithelia.

  4. Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

    PubMed Central

    Asaithamby, Aroumougame; Hu, Burong; Delgado, Oliver; Ding, Liang-Hao; Story, Michael D.; Minna, John D.; Shay, Jerry W.; Chen, David J.

    2011-01-01

    DNA damage and consequent mutations initiate the multistep carcinogenic process. Differentiated cells have a reduced capacity to repair DNA lesions, but the biological impact of unrepaired DNA lesions in differentiated lung epithelial cells is unclear. Here, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We showed, consistent with existing notions that the kinetics of loss of simple double-strand breaks (DSBs) were significantly reduced in organotypic 3D culture compared to kinetics of repair in two-dimensional (2D) culture. Strikingly, we found that, unlike simple DSBs, a majority of complex DNA lesions were irreparable in organotypic 3D culture. Levels of expression of multiple DNA damage repair pathway genes were significantly reduced in the organotypic 3D culture compared with those in 2D culture providing molecular evidence for the defective DNA damage repair in organotypic culture. Further, when differentiated cells with unrepaired DNA lesions re-entered the cell cycle, they manifested a spectrum of gross-chromosomal aberrations in mitosis. Our data suggest that downregulation of multiple DNA repair pathway genes in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis. PMID:21421565

  5. Direct contact between boar spermatozoa and porcine oviductal epithelial cell (OEC) cultures is needed for optimal sperm survival in vitro.

    PubMed

    Yeste, M; Lloyd, R E; Badia, E; Briz, M; Bonet, S; Holt, W V

    2009-07-01

    Oviductal epithelial cell (OEC) co-culture prolongs sperm viability and motility in vitro in a number of species including humans and horses. This study has sought to determine the effects of homologous OEC co-culture on boar sperm function. To determine whether the effects on spermatozoa were specifically caused by co-culture with or by OEC secretions, or by both factors together, a number of co-culture and cell-conditioned medium (CM) experiments were conducted. Firstly, Percoll-washed spermatozoa were co-cultured with OECs and pig kidney epithelial (LLC-PK1) cells, and in medium without cells. Secondly, Percoll-washed spermatozoa were incubated with CM derived from both OECs and LLC-PK1 cells and in unconditioned medium. A number of sperm function parameters were assessed after 5, 30, 60, 90, 120, and 180 min, and 24h of co-culturing or incubation with CM. Of all the sperm function parameters investigated, the percentage (%) viability data yielded the most interesting results. OECs (mean+/-S.E.M.; 31.2+/-1.10) were better than LLC-PK1 cells (24.3+/-0.93) at prolonging the viability of unbound spermatozoa after 24h of co-culturing (P<0.05). Also after 24h, the viability of spermatozoa bound to the OECs (77.6+/-1.83) was significantly higher than in the case of the LLC-PK1 cells (53.5+/-1.43; P<0.001). Other sperm function parameters, e.g., capacitation and motility, were also influenced by OEC co-culturing and incubation with CM, although to a lesser degree. In conclusion, porcine homologous OEC co-culture and CM incubation specifically affect sperm function. However, we propose that it is OEC co-culturing, rather than OEC-CM, that has the greater influence.

  6. Estradiol selectively regulates innate immune function by polarized human uterine epithelial cells in culture

    PubMed Central

    Fahey, JV; Wright, JA; Shen, L; Smith, JM; Ghosh, M; Rossoll, RM; Wira, CR

    2016-01-01

    The goal of this study was to examine the role of E2 in regulating innate immune protection by human uterine epithelial cells (UECs). Recognizing that UECs produce cytokines and chemokines to recruit and activate immune cells as well as viral and bacterial antimicrobials, we sought to examine the effect of E2 on constitutive and Toll-like receptor (TLR) agonist (lipopolysaccharide (LPS) and poly (I:C))-induced immune responses. The secretion by polarized UECs in culture of interleukin (IL)-6, macrophage inhibitory factor (MIF), and secretory leukocyte protease inhibitor (SLPI) was examined as well as the mRNA expression of human β-defensin-2 (HBD2), tumor necrosis factor (TNF)-α, IL-8, and nuclear factor (NF)-kB. When incubated with E2 for 24–48 h, we found that E2 stimulated UEC secretion of SLPI (fourfold) and mRNA expression of HBD2 (fivefold). Moreover, when antibacterial activity in UEC secretions was measured using Staphylococcus aureus, E2 increased the secretion of soluble factor(s) with antibacterial activity. In contrast, E2 had no effect on constitutive secretion of proinflammatory cytokines and chemokines by UECs but completely inhibited LPS- and poly (I:C)-induced secretion of MIF, IL-6, and IL-8. Estradiol also reversed the stimulatory effects of IL-1β on mRNA expression of TNF-α, IL-8, and NF-kB by 85, 95, and 70%, respectively. As SLPI is known to inhibit NF-kB expression, these findings suggest that E2 inhibition of proinflammatory cytokines may be mediated through SLPI regulation of NF-kB. Overall, these findings indicate that the production of cytokines, chemokines, and antimicrobials by UECs are differentially regulated by E2. Further, it suggests that with E2 regulation, epithelial cells that line the uterine cavity have evolved immunologically to be sensitive to viral and bacterial infections as well as the constraints of procreation. PMID:19079193

  7. Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC).

    PubMed

    Nossol, Constanze; Diesing, A-K; Walk, N; Faber-Zuschratter, H; Hartig, R; Post, A; Kluess, J; Rothkötter, H-J; Kahlert, S

    2011-07-01

    The specific function of the epithelium as critical barrier between the intestinal lumen and the organism's internal microenvironment is reflected by permanent maintenance of intercellular junctions and cellular polarity. The intestinal epithelial cells are responsible for absorption of nutritional components, facing mechanical stress and a changing oxygen supplementation via blood stream. Oxygen itself can regulate the barrier and the absorptive function of the epithelium. Therefore, we compared the dish cell culture, the transwell-like membrane culture and the oxygen enriched air-liquid interface (ALI) culture. We demonstrated strong influence of the different culture conditions on morphology and function of intestinal porcine epithelial cell lines in vitro. ALI culture resulted in a significant increase in cell number, epithelial cell layer thickness and expression as well as apical localisation of the microvilli-associated protein villin. Remarkable similarities regarding the morphological parameters were observed between ALI cultures and intestinal epithelial cells in vivo. Furthermore, the functional analysis of protein uptake and degradation by the epithelial cells demonstrated the necessity of sufficient oxygen supply as achieved in ALI cultures. Our study is the first report providing marked evidence that optimised oxygen supply using ALI cultures directly affects the morphological differentiation and functional properties of intestinal epithelial cells in vitro.

  8. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A serum-free, feeder-cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1 wk old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder-cell layers of mit...

  9. Tissue Harvesting Site and Culture Medium Affect Attachment, Growth, and Phenotype of Ex Vivo Expanded Oral Mucosal Epithelial Cells.

    PubMed

    Islam, Rakibul; Eidet, Jon Roger; Badian, Reza A; Lippestad, Marit; Messelt, Edward; Griffith, May; Dartt, Darlene A; Utheim, Tor Paaske

    2017-04-06

    Transplantation of cultured oral mucosal epithelial cells (OMECs) is a promising treatment strategy for limbal stem cell deficiency. In order to improve the culture method, we investigated the effects of four culture media and tissue harvesting sites on explant attachment, growth, and phenotype of OMECs cultured from Sprague-Dawley rats. Neither choice of media or harvesting site impacted the ability of the explants to attach to the culture well. Dulbecco's modified Eagle's medium/Ham's F12 (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI) supported the largest cellular outgrowth. Fold outgrowth was superior from LL explants compared to explants from the buccal mucosa (BM), HP, and transition zone of the lower lip (TZ) after six-day culture. Putative stem cell markers were detected in cultures grown in DMEM and RPMI. In DMEM, cells from TZ showed higher colony-forming efficiency than LL, BM, and HP. In contrast to RPMI, DMEM both expressed the putative stem cell marker Bmi-1 and yielded cell colonies. Our data suggest that OMECs from LL and TZ cultured in DMEM give rise to undifferentiated cells with high growth capacity, and hence are the most promising for treatment of limbal stem cell deficiency.

  10. Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

    PubMed Central

    Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2013-01-01

    Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408

  11. CULTURE CONDITIONS AFFECT HUMAN AIRWAY EPITHELIAL CELL RESPONSE TO DIESEL PARTICLE EXPOSURE IN VITRO

    EPA Science Inventory

    Diesel exhaust particles (DEP) are a ubiquitous ambient air contaminant that may contribute to the health effects of particulate matter inhalation. In vitro studies have shown that DEP exposure induces pro-inflammatory proteins in human airway epithelial cells (HAEC) with varying...

  12. Neutrophil and asbestos fiber-induced cytotoxicity in cultured human mesothelial and bronchial epithelial cells.

    PubMed

    Kinnula, V L; Raivio, K O; Linnainmaa, K; Ekman, A; Klockars, M

    1995-03-01

    This study investigates reactive oxygen species generation and oxidant-related cytotoxicity induced by amosite asbestos fibers and polymorphonuclear leucocytes (PMNs) in human mesothelial cells and human bronchial epithelial cells in vitro. Transformed human pleural mesothelial cells (MET 5A) and bronchial epithelial cells (BEAS 2B) were treated with amosite (2 micrograms/cm2) for 48 h. After 24 h of incubation, the cells were exposed for 1 h to nonactivated or amosite (50 micrograms) activated PMNs, washed, and incubated for another 23 h. Reactive oxygen species generation by the PMNs and the target cells was measured by chemiluminescence. Cell injury was assessed by cellular adenine nucleotide depletion, extracellular release of nucleotides, and lactate dehydrogenase (LDH). Amosite-activated (but also to a lesser degree nonactivated) PMNs released substantial amounts of reactive oxygen metabolites, whereas the chemiluminescence of amosite-exposed mesothelial cells and epithelial cells did not differ from the background. Amosite treatment (48 h) of the target cells did not change intracellular adenine nucleotides (ATP, ADP, AMP) or nucleotide catabolite products (xanthine, hypoxanthine, and uric acid). When the target cells were exposed to nonactivated PMNs, significant adenine nucleotide depletion and nucleotide catabolite accumulation was observed in mesothelial cells only. In separate experiments, when the target cells were exposed to amosite-activated PMNs, the target cell injury was further potentiated compared with the amosite treatment alone or exposure to nonactivated PMNs. In conclusion, this study suggests the importance of inflammatory cell-derived free radicals in the development of amosite-induced mesothelial cell injury.

  13. Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics.

    PubMed

    Lai, Jui-Yang; Ma, David Hui-Kang

    2013-01-01

    Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

  14. In situ-forming click-crosslinked gelatin based hydrogels for 3D culture of thymic epithelial cells.

    PubMed

    Truong, Vinh X; Hun, Michael L; Li, Fanyi; Chidgey, Ann P; Forsythe, John S

    2016-07-21

    Hydrogels prepared from naturally derived gelatin can provide a suitable environment for cell attachment and growth, making them favourable materials in tissue engineering. However, physically crosslinked gelatin hydrogels are not stable under physiological conditions while chemical crosslinking of gelatin by radical polymerization may be harmful to cells. In this study, we attached the norbornene functional group to gelatin, which was subsequently crosslinked with a polyethylene glycol (PEG) linker via the nitrile oxide-norbornene click reaction. The rapid crosslinking process allows the hydrogel to be formed within minutes of mixing the polymer solutions under physiological conditions, allowing the gels to be used as injectable materials. The hydrogels properties including mechanical strength, swelling and degradation, can be tuned by changing either the ratio of the reacting groups or the total concentration of the polymer precursors. Murine embryonic fibroblastic cells cultured in soft gels (2 wt% of gelatin and 1 wt% of PEG linker) demonstrated high cell viability as well as similar phenotypic profiles (PDGFRα and MTS15) to Matrigel cultures over 5 days. Thymic epithelial cell and fibroblast co-cultures produced epithelial colonies in these gels following 7 days incubation. These studies demonstrate that gelatin based hydrogels, prepared using "click" crosslinking, provide a robust cell culture platform with retained benefits of the gelatin material, and are therefore suitable for use in various tissue engineering applications.

  15. Primary airway epithelial cell culture and asthma in children-lessons learnt and yet to come.

    PubMed

    McLellan, Kirsty; Shields, Mike; Power, Ultan; Turner, Steve

    2015-12-01

    Until recently the airway epithelial cell (AEC) was considered a simple barrier that prevented entry of inhaled matter into the lung parenchyma. The AEC is now recognized as having an important role in the inflammatory response of the respiratory system to inhaled exposures, and abnormalities of these responses are thought to be important to asthma pathogenesis. This review first explores how the challenges of studying nasal and bronchial AECs in children have been addressed and then summarizes the results of studies of primary AEC function in children with and without asthma. There is good evidence that nasal AECs may be a suitable surrogate for the study of certain aspects of bronchial AEC function, although bronchial AECs remain the gold standard for asthma research. There are consistent differences between children with and without asthma for nasal and bronchial AEC mediator release following exposure to a range of pro-inflammatory stimulants including interleukins (IL)-1β, IL-4, and IL-13. However, there are inconsistencies between studies, e.g., release of IL-6, an important pro-inflammatory cytokine, is not increased in children with asthma relative to controls in all studies. Future work should expand current understanding of the "upstream" signalling pathways in AEC, study AEC from children before the onset of asthma symptoms and in vitro models should be developed that replicate the in vivo status more completely, e.g., co-culture with dendritic cells. AECs are difficult to obtain from children and collaboration between centers is expected to yield meaningful advances in asthma understanding and ultimately help deliver novel therapies.

  16. Increased ROS production in non-polarized mammary epithelial cells induces monocyte infiltration in 3D culture.

    PubMed

    Li, Linzhang; Chen, Jie; Xiong, Gaofeng; St Clair, Daret K; Xu, Wei; Xu, Ren

    2017-01-01

    Loss of epithelial cell polarity promotes cell invasion and cancer dissemination. Therefore, identification of factors that disrupt polarized acinar formation is crucial. Reactive oxygen species (ROS) drive cancer progression and promote inflammation. Here, we show that the non-polarized breast cancer cell line T4-2 generates significantly higher ROS levels than polarized S1 and T4R cells in three-dimensional (3D) culture, accompanied by induction of the nuclear factor κB (NF-κB) pathway and cytokine expression. Minimizing ROS in T4-2 cells with antioxidants reestablished basal polarity and inhibited cell proliferation. Introducing constitutively activated RAC1 disrupted cell polarity and increased ROS levels, indicating that RAC1 is a crucial regulator that links cell polarity and ROS generation. We also linked monocyte infiltration with disruption of polarized acinar structure using a 3D co-culture system. Gain- and loss-of-function experiments demonstrated that increased ROS in non-polarized cells is necessary and sufficient to enhance monocyte recruitment. ROS also induced cytokine expression and NF-κB activity. These results suggest that increased ROS production in mammary epithelial cell leads to disruption of cell polarity and promotes monocyte infiltration.

  17. Azithromycin induces anti-viral effects in cultured bronchial epithelial cells from COPD patients

    PubMed Central

    Menzel, Mandy; Akbarshahi, Hamid; Bjermer, Leif; Uller, Lena

    2016-01-01

    Rhinovirus infection is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations and may contribute to the development into severe stages of COPD. The macrolide antibiotic azithromycin may exert anti-viral actions and has been reported to reduce exacerbations in COPD. However, little is known about its anti-viral actions on bronchial epithelial cells at clinically relevant concentrations. Primary bronchial epithelial cells from COPD donors and healthy individuals were treated continuously with azithromycin starting 24 h before infection with rhinovirus RV16. Expression of interferons, RIG-I like helicases, pro-inflammatory cytokines and viral load were analysed. Azithromycin transiently increased expression of IFNβ and IFNλ1 and RIG-I like helicases in un-infected COPD cells. Further, azithromycin augmented RV16-induced expression of interferons and RIG-I like helicases in COPD cells but not in healthy epithelial cells. Azithromycin also decreased viral load. However, it only modestly altered RV16-induced pro-inflammatory cytokine expression. Adding budesonide did not reduce interferon-inducing effects of azithromycin. Possibly by inducing expression of RIG-I like helicases, azithromycin increased rhinovirus-induced expression of interferons in COPD but not in healthy bronchial epithelium. These effects would reduce bronchial viral load, supporting azithromycin’s emerging role in prevention of exacerbations of COPD. PMID:27350308

  18. Protection against radiation-induced oxidative stress in cultured human epithelial cells by treatment with antioxidant agents

    SciTech Connect

    Wan, X. Steven; Ware, Jeffrey H.; Zhou, Zhaozong; Donahue, Jeremiah J.; Guan, Jun; Kennedy, Ann R. . E-mail: akennedy@mail.med.upenn.edu

    2006-04-01

    Purpose: To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells. Methods and Materials: The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, {alpha}-lipoic acid, L-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, {gamma}-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay. Results: The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure. Conclusion: The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects.

  19. Efficient protection by cationized catalase against H2O2 injury in primary cultured alveolar epithelial cells.

    PubMed

    Nemoto, Takayuki; Kawakami, Shigeru; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2007-08-16

    Increasing evidence suggests that hydrogen peroxide plays an important role in alveolar epithelial injury produced during many inflammatory lung diseases. In this study, the successful prevention of hydrogen peroxide (H(2)O(2))-induced injury in primary cultured rabbit alveolar epithelial cells by cationized catalase is described. Cationized catalase was synthesized by direct chemical modification to enhance its association with alveolar epithelial cells. Cationized catalase exhibited a 22.3-fold higher cellular association at 2 h than native catalase, and incubation of cationized catalase with the cells produced a 2.19-fold intracellular catalase activity, which suggested that cationized catalase distributed both to the cell membrane and into the cell interior. Cationized catalase markedly suppressed H(2)O(2)-induced cell injury. In addition, electron spin resonance spectrometry analysis revealed that cationized catalase effectively eliminated H(2)O(2) produced in the medium by glucose plus glucose oxidase. On the other hand, polyethylene glycol-modified catalase (PEG-catalase) did not have any protective effect against H(2)O(2)-induced cell injury although PEG-catalase exhibited a 2.49-fold higher cellular association at 2 h than native catalase. These results suggest that cationization of catalase is a promising strategy for the treatment of many of inflammatory lung diseases.

  20. Evaluation of low-dosage environmental mutagens with a long-term, cultured epithelial cell line

    SciTech Connect

    Li, C.S.; Lin, R.H.

    1996-12-31

    Polycyclic or polynuclear aromatic hydrocarbons (PAHs) are a group of compounds consisting of at least two fused aromatic rings and are common environmental contaminants in soil, water, and air. Additionally, some PAHs are considered to be mutagenic, carcinogenic, and cytotoxic compounds. BaA (Benz(a)anthracene) and BaP (Benzo(a)pyrene) are the two major unsubstituted PAHs identified by the USEPA as priority pollutants. BaA is suspected to be a human carcinogen by the US Department of Health and Human Services, whereas BaP is regarded as an animal carcinogen. It is estimated that the emission concentrations of BaA and BaP from mainstream cigarette smoke are 20-70 ng per g cigarette and 20-40 ng per g cigarette, respectively, with an average sidestream to mainstream weight ratio of 3. In addition, BaA and BaP are also the main emissions from diesel particulate extracts with mean concentrations of 500 ppm and 40 ppm, respectively. From a recent report concerning PAHs in urban areas all over the world, the airborne concentration of BaP was typically found to be in the range of 1-20 ng/m{sup 3} in Europe and 1 ng/m{sup 3} in the U.S.A. For BaA, the concentrations commonly ranged from I to 50 ng/m{sup 3} in Europe and from 0.1 to 1 ng/m{sup 3} in the U.S.A. Therefore, the long term exposure to these two compounds may cause health effects. This study examines long-term cultured epithelial cell lines exposed to BaP or BaA, in vitro, and the frequency of HGPRT mutants. 19 refs., 3 figs.

  1. GLUCOSE METABOLITE PATTERNS AS MARKERS OF FUNCTIONAL DIFFERENTIATION IN FRESHLY ISOLATED AND CULTURED MOUSE MAMMARY EPITHELIAL CELLS

    SciTech Connect

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

    1980-06-01

    In the mammary gland of nonruminant animals, glucose is utilized in a characteristic and unique way during lactation. We have measured the incorporation of glucose carbon from [U-{sup 14}C] glucose into intermediary metabolites and metabolic products in mammary epithelial cells from virgin, pregnant, and lactating mice and demonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate were important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells from pregnant mice have a pattern similar to freshly isolated cells from pregnant mice. The pattern of cells from lactating mice is different from that of the cells of origin, and resembles that of the cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  2. Propagation of normal human epithelial cell populations using an in vivo culture system. Description and applications.

    PubMed Central

    Klein-Szanto, A. J.; Terzaghi, M.; Mirkin, L. D.; Martin, D.; Shiba, M.

    1982-01-01

    A new model using xenotransplanted human epithelia was developed for the study of toxic and carcinogenic effects of chemicals. Epithelial cells from the respiratory tract of 4 male and 3 female premature and fullterm fetuses were enzymatically removed and inoculated into deepithelialized rat tracheas. These were sealed at both ends and transplanted subcutaneously into nude mice. After 3-4 weeks, a normal mucociliary epithelium covered the tracheal lumen. At this stage the epithelial cells could be isolated again and transplanted into new denuded rat tracheas. This passaging could be repeated up to six times, each permitting an amplification factor of approximately 3. Tracheal transplants containing cells of human origin (in vivo Passages 2-4) were treated with 7,12-dimethylbenz(a)anthracene. Hyperplasias, squamous metaplasias, and dysplasias were seen 1-8 weeks after initiation of treatment, indicating that the responses of human and rodent epithelial cells to polycyclic aromatic hydrocarbons are similar. Initial experiments with skin and esophageal epithelia suggest that other covering epithelia could also be used in this fashion for evaluation of toxicants and carcinogens that are likely to come into contact with these tissues. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:6821529

  3. N-glycosylated proteins are involved in efficient internalization of Klebsiella pneumoniae by cultured human epithelial cells.

    PubMed Central

    Fumagalli, O; Tall, B D; Schipper, C; Oelschlaeger, T A

    1997-01-01

    Klebsiella pneumoniae obtained from patients with urinary tract infections is able to invade cultured human epithelial cells. The internalization process is dependent upon both microfilaments and microtubules. To better understand the interaction of these invasive bacteria with the host cell receptor(s), bladder, lung, and ileocecal epithelial cells were infected with K. pneumoniae in the presence of various lectins possessing multiple glycan specificities. It was found that the N-acetylglucosamine (GlcNAc)-specific lectins concanavalin A, Datura stramonium agglutinin, and wheat germ agglutinin significantly inhibited the invasion of K. pneumoniae into these cells but did not interfere with the internalization of an invasive strain of Salmonella typhimurium. Conversely, internalization of K. pneumoniae but not S. typhimurium was also significantly inhibited when the bacteria were pretreated with GlcNAc or chitin hydrolysate, a GlcNAc polymer, prior to the gentamicin invasion assay. Other carbohydrates such as glucose, galactose, mannose, fucose, and N-acetylneuraminic acid had no inhibitory effects on K. pneumoniae uptake. Furthermore, internalization of K. pneumoniae but not S. typhimurium by HCT8 cells was also significantly inhibited when eukaryotic protein glycosylation was interrupted by tunicamycin or when host N-linked surface glycans were removed by pretreatment with N-glycosidase F. These studies suggest that a N-glycosylated protein receptor is involved in the internalization of K. pneumoniae by human epithelial cells in vitro. The results also indicate that internal GlcNAc residues might be a carbohydrate component of the receptor. PMID:9353018

  4. Effect of cigarette smoke extract on P-glycoprotein function in primary cultured and newly developed alveolar epithelial cells.

    PubMed

    Takano, Mikihisa; Naka, Ryosuke; Sasaki, Yoshihiro; Nishimoto, Saori; Yumoto, Ryoko

    2016-12-01

    The effect of cigarette smoke extract (CSE) on P-glycoprotein (P-gp) function in the distal lung is unclear. In this study, we first examined the expression and function of P-gp and the effect of CSE in rat primary cultured alveolar epithelial cells. The expression of P-gp protein was observed in type I-like cells, but not in type II cells. In type I-like cells, rhodamine 123 (Rho123) accumulation was enhanced by various P-gp inhibitors such as verapamil and cyclosporine A. In addition, the expression of P-gp mRNAs, mdr1a and mdr1b, as well as P-gp activity increased along with the transdifferentiation. When type I-like cells were co-incubated with CSE, P-gp activity was suppressed. Next, we attempted to clarify the effect of CSE on P-gp function in human-derived cultured alveolar epithelial cells. For this purpose, we isolated an A549 clone (A549/P-gp) expressing P-gp, because P-gp expression in native A549 cells was negligible. In A549/P-gp cells, P-gp was functionally expressed, and the inhibitory effect of CSE on P-gp was observed. These results suggested that smoking would directly suppress P-gp activity, and that A549/P-gp cell line should be a useful model to further study the effect of xenobiotics on P-gp function in the alveolar epithelial cells.

  5. Morphology, behavior, and interaction of cultured epithelial cells after the antibody-induced disruption of keratin filament organization

    PubMed Central

    1983-01-01

    The organization of intermediate filaments in cultured epithelial cells was rapidly and radically affected by intracellularly injected monoclonal antikeratin filament antibodies. Different antibodies had different effects, ranging from an apparent splaying apart of keratin filament bundles to the complete disruption of the keratin filament network. Antibodies were detectable within cells for more than four days after injection. The antibody-induced disruption of keratin filament organization had no light-microscopically discernible effect on microfilament or microtubule organization, cellular morphology, mitosis, the integrity of epithelial sheets, mitotic rate, or cellular reintegration after mitosis. Cell-to-cell adhesion junctions survived keratin filament disruption. However, antibody injected into a keratinocyte-derived cell line, rich in desmosomes, brought on a superfasciculation of keratin filament bundles, which appeared to pull desmosomal junctions together, suggesting that desmosomes can move in the plane of the plasma membrane and may only be 'fixed' by their anchoring to the cytoplasmic filament network. Our observations suggest that keratin filaments are not involved in the establishment or maintenance of cell shape in cultured cells. PMID:6187752

  6. Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture.

    PubMed

    Jorgens, Danielle M; Inman, Jamie L; Wojcik, Michal; Robertson, Claire; Palsdottir, Hildur; Tsai, Wen-Ting; Huang, Haina; Bruni-Cardoso, Alexandre; López, Claudia S; Bissell, Mina J; Xu, Ke; Auer, Manfred

    2017-01-01

    The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.

  7. Two-dimensional culture of human pancreatic adenocarcinoma cells results in an irreversible transition from epithelial to mesenchymal phenotype.

    PubMed

    Kang, Ya'an; Zhang, Ran; Suzuki, Rei; Li, Shao-qiang; Roife, David; Truty, Mark J; Chatterjee, Deyali; Thomas, Ryan M; Cardwell, James; Wang, Yu; Wang, Huamin; Katz, Matthew H; Fleming, Jason B

    2015-02-01

    Many commercially available cell lines have been in culture for ages, acquiring phenotypes that differ from the original cancers from which these cell lines were derived. Therefore, research on new cell lines could improve the success rates of translational research in cancer. We have developed methods for the isolation and culture of human pancreatic ductal adenocarcinoma (PDAC) cells from murine xenografts of human PDAC. We hypothesize that phenotypes of PDAC cells are modified by in vitro culture conditions over time and by in vivo implantation. Patient-derived xenografts were created in immunodeficient mice using surgically resected tumor specimens. These murine xenografts were then used to establish human PDAC cell lines in culture. Earlier (<5) passage and later (>20) passage cell lines were evaluated separately regarding proliferation, cell cycle, genetic mutations, invasiveness, chemosensitivity, tumorigenesis, epithelial-mesenchymal transition (EMT) status, and proteomics. Later passage cells accelerated their doubling time and colony formation, and were more concentrated in the G0/G1 phase and less in the G2/M checkpoint phase. Later passage cells were more sensitive to gemcitabine and 5-fluorouracil than earlier passage cells, but all four new cell lines were more chemo-resistant compared with commercial ATCC cell lines. EMT induction was observed when establishing and passaging cell lines in vitro and furthermore by growing them as subcutaneous tumors in vivo. This study demonstrates a novel approach to the establishment of PDAC cell lines and observes a process by which newly established cell lines undergo phenotypic changes during in vitro culture and in vivo tumorigenesis. This may help explain differences of treatment effects often observed between experiments conducted in vitro, in vivo, and in human clinical trials.

  8. Glucocorticoid receptors in primary cultures of mouse mammary epithelial cells: characterization and modulation by prolactin and cortisol

    SciTech Connect

    Schneider, W.; Shyamala, G.

    1985-06-01

    Mammary epithelial cells isolated from midpregant mice and cultured on collagen gels contain soluble glucocorticoid receptors. The kinetics of binding of dexamethasone reveal a saturable binding site (dissociation constant (K /sub d/), approximately 1 nM), and the binding site obeys a steroid specificity characteristic of a glucocorticoid receptor. As with the receptor isolated from intact glands, the receptor from the cultured cells also requires the addition of dithiothreitol for maximal binding of dexamethasone. The receptors are maintained at in vivo levels (approximately 1.3 pmol/mg DNA) for at least a period of 10 days in culture. However, the presence of both cortisol and PRL is required for the maintenance of the receptors, and the effect of both these hormones is dose dependent.

  9. Harvesting Human Prostate Tissue Material and Culturing Primary Prostate Epithelial Cells.

    PubMed

    Frame, Fiona M; Pellacani, Davide; Collins, Anne T; Maitland, Norman J

    2016-01-01

    In order to fully explore the biology of a complex solid tumor such as prostate cancer, it is desirable to work with patient tissue. Only by working with cells from a tissue can we take into account patient variability and tumor heterogeneity. Cell lines have long been regarded as the workhorse of cancer research and it could be argued that they are of most use when considered within a panel of cell lines, thus taking into account specified mutations and variations in phenotype between different cell lines. However, often very different results are obtained when comparing cell lines to primary cells cultured from tissue. It stands to reason that cells cultured from patient tissue represents a close-to-patient model that should and does produce clinically relevant data. This chapter aims to illustrate the methods of processing, storing and culturing cells from prostate tissue, with a description of potential uses.

  10. Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

    SciTech Connect

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

    1981-01-01

    In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from (U-/sup 14/C)glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  11. Medium Calcium Concentration Determines Keratin Intermediate Filament Density and Distribution in Immortalized Cultured Thymic Epithelial Cells (TECs)

    NASA Astrophysics Data System (ADS)

    Sands, Sandra S.; Meek, William D.; Hayashi, Jun; Ketchum, Robert J.

    2005-08-01

    Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4 5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37°C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 [mu]g/ml), transferrin (10 [mu]g/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

  12. Effects of species and cellular activity of oviductal epithelial cells on their dialogue with co-cultured mouse embryos.

    PubMed

    Tan, Xiu-Wen; Ma, Suo-Feng; Yu, Jian-Ning; Zhang, Xia; Lan, Guo-Cheng; Liu, Xin-Yong; Han, Zheng-Bin; Tan, Jing-He

    2007-01-01

    An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because

  13. Infection of cultured intestinal epithelial cells with severe acute respiratory syndrome coronavirus.

    PubMed

    Cinatl, J; Hoever, G; Morgenstern, B; Preiser, W; Vogel, J-U; Hofmann, W-K; Bauer, G; Michaelis, M; Rabenau, H F; Doerr, H W

    2004-08-01

    To identify a model for the study of intestinal pathogenesis of severe acute respiratory syndrome (SARS) we tested the sensitivity of six human intestinal epithelial cell lines to infection with SARS coronavirus (SARS-CoV). In permissive cell lines, effects of SARS-CoV on cellular gene expression were analysed using high-density oligonucleotide arrays. Caco-2 and CL-14 cell lines were found to be highly permissive to SARS-CoV, due to the presence of angiotensin-converting enzyme 2 as a functional receptor. In both cell lines, SARS-CoV infection deregulated expression of cellular genes which may be important for the intestinal pathogenesis of SARS.

  14. The role of proteases in fibronectin matrix remodeling in thyroid epithelial cell monolayer cultures.

    PubMed

    Nezi, Luigi; Greco, Dario; Nitsch, Lucio; Garbi, Corrado

    2002-01-01

    Fischer rat thyroid (FRT) cells organize a matrix of extracellular fibronectin (FN) fibrils, which undergoes extensive remodeling according to cell culture confluence. In non-confluent cells FN forms a fibrillar array associated with the ventral cell surface. However, basal FN is progressively removed in confluent cultures and substituted by non-fibrillar FN deposits at lateral cell domains in regions of cell-cell contacts. FRT cells secrete and expose on the plasma membrane the tissue-type plasminogen activator and, in serum-free cultures, plasminogen induces a rapid loss of FN fibrils. Incubation with plasmin inhibitors greatly reduces this effect. FRT cells also express annexin II, a plasminogen receptor, suggesting that plasmin activity is associated with the pericellular enviroment. This is in agreement with the observation that a great reduction in FN degradation is observed if the cells are pre-incubated with carboxypeptidase B, which prevents plasminogen binding to the cells. A gelatinolytic activity with a molecular weigth equivalent to MMP-2 has been demonstrated by zymography of culture media, and the presence of MMP-2 and MT1-MMP on the cell plasma membrane has been detected by immunofluorescence. These results indicate that in the FN remodeling process, occurring during FRT epithelium maturation, both plasmin-dependent (tPA activated) and plasmin-independent proteolytic activities are involved.

  15. Povidone-iodine-induced cell death in cultured human epithelial HeLa cells and rat oral mucosal tissue.

    PubMed

    Sato, So; Miyake, Masao; Hazama, Akihiro; Omori, Koichi

    2014-07-01

    Although povidone-iodine (PVP-I) has been used as a gargle since 1956, its effectiveness and material safety have been remained controversial. The aim of this study was to investigate the toxicity of PVP-I to epithelial cells in a concentration range significantly lower than that used clinically. Study design was in vitro laboratory investigations and in vivo histological and immunologic analysis. We examined the effects of PVP-I at concentrations of 1 × 10(-2) to 1 × 10(3) μM and 1 × 10(-4) to 1 × 10 μM on HeLa cells as a model of epithelial cells and rat oral mucosa, respectively, after 1 or 2 days of exposure. Annexin V/FLUOS was used to distinguish live, apoptotic and necrotic cells. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method was also used to observe whether apoptotic epithelial cells exist in rat oral mucosa after 1 day of exposure of PVP-I. HeLa cells developed concentration-dependent cytotoxicity, and epithelium of rat oral mucosa was thinned in a concentration-dependent manner. HeLa cell apoptosis increased after 1 × 10(0) μM of PVP-I exposure for 2 days. In the TUNEL method, many apoptotic epithelial cells were observed in the rat oral mucosa after 1 day of exposure to diluted 1 × 10(-2) μM of PVP-I, but minimal apoptotic epithelial cells were observed using 1 × 10(-3) μM of PVP-I. Our findings suggest that exposure to PVP-I, of which concentrations are even lower than those used clinically, causes toxicity in epithelial cells. This knowledge would help us better understand the risk of the use of PVP-I against mucosa.

  16. Transcriptome Analysis of Cultured Limbal Epithelial Cells on an Intact Amniotic Membrane following Hypothermic Storage in Optisol-GS

    PubMed Central

    Paaske Utheim, Tor; Salvanos, Panagiotis; Aass Utheim, Øygunn; Ræder, Sten; Pasovic, Lara; Olstad, Ole Kristoffer; Fideliz de la Paz, Maria; Sehic, Amer

    2016-01-01

    The aim of the present study was to investigate the molecular mechanisms underlying activation of cell death pathways using genome-wide transcriptional analysis in human limbal epithelial cell (HLEC) cultures following conventional hypothermic storage in Optisol-GS. Three-week HLEC cultures were stored in Optisol-GS for 2, 4, and 7 days at 4 °C. Partek Genomics Suite software v.6.15.0422, (Partec Inc., St. Louis, MO, USA) was used to identify genes that showed significantly different (P < 0.05) levels of expression following hypothermic storage compared to non-stored cell sheets. There were few changes in gene expression after 2 days of storage, but several genes were differently regulated following 4 and 7 days of storage. The histone-coding genes HIST1H3A and HIST4H4 were among the most upregulated genes following 4 and 7 days of hypothermic storage. Bioinformatic analysis suggested that these two genes are involved in a functional network highly associated with cell death, necrosis, and transcription of RNA. HDAC1, encoding histone deacetylase 1, was the most downregulated gene after 7 days of storage. Together with other downregulated genes, it is suggested that HDAC1 is involved in a regulating network significantly associated with cellular function and maintenance, differentiation of cells, and DNA repair. Our data suggest that the upregulated expression of histone-coding genes together with downregulated genes affecting cell differentiation and DNA repair may be responsible for increased cell death following hypothermic storage of cultured HLEC. In summary, our results demonstrated that a higher number of genes changed with increasing storage time. Moreover, in general, larger differences in absolute gene expression values were observed with increasing storage time. Further understanding of these molecular mechanisms is important for optimization of storage technology for limbal epithelial sheets. PMID:26901233

  17. Lactobacillus acidophilus binds to MUC3 component of cultured intestinal epithelial cells with highest affinity.

    PubMed

    Das, Jugal Kishore; Mahapatra, Rajani Kanta; Patro, Shubhransu; Goswami, Chandan; Suar, Mrutyunjay

    2016-04-01

    Lactobacillus strains have been shown to adhere to the mucosal components of intestinal epithelial cells. However, established in vitro adhesion assays have several drawbacks in assessing the adhesion of new Lactobacillus strains. The present study aimed to compare the adhesion of four different Lactobacillus strains and select the most adherent microbe, based on in silico approach supported by in vitro results. The mucus-binding proteins in Lactobacillus acidophilus, L. plantarum, L. brevis and L. fermentum were identified and their capacities to interact with intestinal mucin were compared by molecular docking analysis. Lactobacillus acidophilus had the maximal affinity of binding to mucin with predicted free energy of -6.066 kcal mol(-1) Further, in vitro experimental assay of adhesion was performed to validate the in silico results. The adhesion of L. acidophilus to mucous secreting colon epithelial HT-29 MTX cells was highest at 12%, and it formed biofilm with maximum depth (Z = 84 μm). Lactobacillus acidophilus was determined to be the most adherent strain in the study. All the Lactobacillus strains tested in this study, displayed maximum affinity of binding to MUC3 component of mucus as compared to other gastrointestinal mucins. These findings may have importance in the design of probiotics and health care management.

  18. Effect of Cryopreserved Amniotic Membrane Orientation on the Expression of Limbal Mesenchymal and Epithelial Stem Cell Markers in Prolonged Limbal Explant Cultures

    PubMed Central

    Lužnik, Zala; Hawlina, Marko; Maličev, Elvira; Bertolin, Marina; Kopitar, Andreja Nataša; Ihan, Alojz; Ferrari, Stefano; Schollmayer, Petra

    2016-01-01

    Purpose To evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC). Methods Limbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67. Results Morphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM. Conclusion Limbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67. PMID:27723792

  19. Ultraviolet microbeam irradiations of cultured newt lung epithelial cells during mitosis

    SciTech Connect

    Cypher, C.

    1983-01-01

    The mechanism of chromosome movement is unknown. The structural environment for this movement is a birefringent, spindle-shaped array of microtubules. Microbeams of ultraviolet light were used to disrupt the mitotic spindles of newt lung epithelial cells to localize force production within spindles and to evaluate the role of microtubules in force generation or transduction. Time-lapse cinephotomicrographic records of cells were made using phase and polarization microscopy. Irradiation effects were correlated with spindle microtubule structure by immuno-gold antitubulin staining. The results demonstrate the pervasive effects of local irradiations upon spindle structure. The spindle compaction observed after irradiations suggests that each half-spindle is under a compressive force. Since the irradiations locally disassemble microtubules, the results suggest that the birefringent microtubules oppose this compressive force.

  20. Cancer Risk-Assessment of Radiation Damage in Ataxia Telangiectasia Heterozygous Human Breast Epithelial Cell Cultures

    NASA Technical Reports Server (NTRS)

    Applewhite, Lisa C.

    2002-01-01

    This paper describes the study of the markers of cellular changes that are found during the onset of carcinogenesis. Several of the biological factors are markers of stress response, oncoprotein expression, and differentiation factors. Oxidative stress response agents such as heat shock proteins (HSPs) protect cells from oxidative stresses such as ionizing radiation. The onocoprotein HER-2/neu, a specific breast cancer marker, indicates early onset of cancer. Additional structural and morphogenetic markers of differentiation were considered in order to determine initial cellular changes at the initial onset of cancer. As an additional consideration, all-trans retinoic acid (RA), a differentiation agent, was considered because of its known role in regulating normal differentiation and inhibiting tumor proliferation via specific nuclear receptors. This paper discusses study and results of the preliminary analyses of gamma irradiation of AT heterozygous human breast epithelial cells (WH). Comparisons are also made of the effects various RA concentrations post-irradiation.

  1. Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy.

    PubMed

    Grabinger, T; Luks, L; Kostadinova, F; Zimberlin, C; Medema, J P; Leist, M; Brunner, T

    2014-05-15

    Intestinal epithelial cells (IECs) not only have a critical function in the absorption of nutrients, but also act as a physical barrier between our body and the outside world. Damage and death of the epithelial cells lead to the breakdown of this barrier function and inflammation due to access of the immune system to compounds of the intestinal flora. Intestinal epithelial damage is frequently associated with various inflammatory disorders, chemo- and radiotherapy as well as drug-mediated toxicity. Until recently, intestinal epithelial-damaging activities of drugs and treatments could be tested only in vivo in animal models because of the poor survival rate of primary IECs ex vivo. The three-dimensional culture and outgrowth of intestinal crypt stem cells into organoids have offered new possibilities to culture and study IECs ex vivo. Here we demonstrate that intestinal organoids are a useful and physiologically relevant model system to study cell death and survival in IECs. We further describe a number of microscopy-based as well as colorimetric methods to monitor and score survival and death of intestinal organoids. Finally, the comparison of organoids isolated from gene-deficient mice and wild-type mice allows investigating the role of specific genes in the regulation of IEC death. Owing to their comparable structure and behavior, intestinal organoids may serve as an interesting and physiologically relevant surrogate system for large- and mid-scale in vitro testing of intestinal epithelium-damaging drugs and toxins, and for the investigation of cell death pathways.

  2. Bovine milk RNases modulate pro-inflammatory responses induced by nucleic acids in cultured immune and epithelial cells.

    PubMed

    Gupta, Sandeep K; Haigh, Brendan J; Seyfert, Hans-Martin; Griffin, Frank J; Wheeler, Thomas T

    2017-03-01

    Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.

  3. High- and low-LET Radiation-induced Chromosome Aberrations in Human Epithelial Cells Cultured in 3-dimensional Matrices

    NASA Technical Reports Server (NTRS)

    Hada, M.; George K.; Cucinotta, F. A.; Wu, H.

    2008-01-01

    Energetic heavy ions pose a great health risk to astronauts who participate in extended ISS missions and will be an even greater concern for future manned lunar and Mars missions. High-LET heavy ions are particularly effective in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, which can be utilized as a biomarker for radiation insults. Previously, we had studied low- and high-LET radiation-induced chromosome aberrations in human epithelial cells cultured in 2-dimension (2D) using the multicolor banding fluorescence in situ hybridization (mBAND) technique. However, it has been realized that the biological response to radiation insult in a 2D in vitro cellular environment can differ significantly from the response in 3-dimension (3D) or at the actual tissue level. In this study, we cultured human epithelial cells in 3D to provide a more suitable model for human tissue. Human mammary epithelial cells (CH184B5F5/M10) were grown in Matrigel to form 3D structures, and exposed to Fe-ions at NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory or 137Cs-gamma radiation source at the University of Texas MD Anderson Cancer Center. After exposure, cells were allowed to repair for 16hr before dissociation and subcultured at low density in 2D. G2 and metaphase chromosomes in the first cell cycle were collected in the first cell cycle after irradiation using a chemical-induced premature chromosome condensation (PCC) technique, and chromosome aberrations were analyzed using mBAND technique. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of interchromosomal aberrations (translocation to unpainted chromosomes) and intrachromosomal aberrations (inversions and deletions within a single painted chromosome). Our data indicate a significant difference in the

  4. Phagosomal pH and glass fiber dissolution in cultured nasal epithelial cells and alveolar macrophages: a preliminary study.

    PubMed Central

    Johnson, N F

    1994-01-01

    The dissolution rate of glass fibers has been shown to be pH sensitive using in vitro lung fluid simulant models. The current study investigated whether there is a difference in phagosomal pH (ppH) between rat alveolar macrophages (AM) and rat nasal epithelial cells (RNEC) and whether such a difference would influence the dissolution of glass fibers. The ppH was measured in cultured AM and RNEC using flow cytometric, fluorescence-emission rationing techniques with fluorescein-labeled, amorphous silica particles. Glass fiber dissolution was determined in AM and RNEC cultured for 3 weeks with fast dissolving glass fibers (GF-A) or slow dissolving ones (GF-B). The mean diameters of GF-A were 2.7 microns and of GF-B, 2.6 microns, the average length of both fibers was approximately 22 to 25 microns. Dissolution was monitored by measuring the length and diameter of intracellular fibers and estimating the volume, assuming a cylindrical morphology. The ppH of AM was 5.2 to 5.8, and the ppH of RNEC was 7.0 to 7.5. The GF-A dissolved more slowly in RNEC than in AM, and no dissolution was evident in either cell type with GF-B. The volume loss with GF-A after a 3-week culture with AM was 66% compared to 45% for cultured RNEC. These results are different from those obtained using in vitro lung fluid-simulant models where dissolution is faster at higher pH. This difference suggests that dissolution rates of glass fibers in AM should not be applied to the dissolution of fibers in epithelial cells. Images Figure 1. a Figure 1. b Figure 2. a Figure 2. b Figure 3. a Figure 3. b PMID:7882965

  5. The majority of enteroaggregative Escherichia coli strains produce the E. coli common pilus when adhering to cultured epithelial cells.

    PubMed

    Avelino, Fabiola; Saldaña, Zeus; Islam, Sohidul; Monteiro-Neto, Valerio; Dall'Agnol, Monique; Eslava, Carlos A; Girón, Jorge A

    2010-11-01

    Enteroaggregative Escherichia coli (EAEC) have emerged as a significant worldwide cause of chronic diarrhea in the pediatric population and in HIV patients. The vast majority of EAEC strains do not produce the aggregative adherence fimbriae I-III (AAFs) so far reported and thus, what adherence factors are present in these strains remains unknown. Here, we investigated the prevalence of the chromosomal E. coli common pilus (ECP) genes and ECP production amongst 130 EAEC strains of diverse origin as well as the role of ECP in EAEC adherence. Through multiplex PCR analysis we found that 96% of EAEC strains contained the ecpA structural pilin gene whereas only 3.1% and 5.4% were positive for AAF fimbrial genes aggA or aafA, respectively. Among the ecpA(+) strains, 63% produced ECP when adhering to cultured epithelial cells. An ecpA mutant derived from prototypic strain 042 (AAF/II(+)) was not altered in adherence suggesting that the AAF/II, and not ECP, plays a major role in this strain. In contrast, strain 278-1 (AAF(-)) deleted of the ecpA gene was significantly reduced in adherence to cultured epithelial cells. In all, these data indicate a potential role of ECP in adherence for EAEC strains lacking the known AAFs and that in association with other adhesive determinants, ECP may contribute to their survival and persistence within the host and in the environment.

  6. Specificity in calcium oxalate adherence to papillary epithelial cells in culture

    SciTech Connect

    Riese, R.J.; Riese, J.W.; Kleinman, J.G.; Wiessner, J.H.; Mandel, G.S.; Mandel, N.S. )

    1988-11-01

    Attachment of microcystallites to cellular membranes may be an important component of the pathophysiology of many diseases including urolithiasis. This study attempts to characterize the interaction of calcium oxalate (CaOx) crystals and apatite (AP) crystals with renal papillary collecting tubule (RPCT) cells in primary culture. Primary cultures of RPCT cells showed the characteristic monolayer growth with sporadically interspersed clumped cells. Cultures were incubated with ({sup 14}C)CaOx crystals, and the crystals that bound were quantified by microscopy and adherent radioactivity. Per unit of cross-sectional area, 32 times more CaOx crystals were bound to the clumps than to the monolayer. CaOx adherence demonstrated concentration-dependent saturation with a {beta} value (fraction of cell culture area binding CaOx crystals) of 0.179 and a 1/{alpha}{sub ox} value of 287 {mu}g/cm{sup 2}. On incubation with AP crystals, CaOx binding demonstrated concentration-dependent inhibition with a 1/{alpha}{sub AP} value of 93 {mu}g/cm{sup 2}. Microcystallite adherence to RPCT cells demonstrates selectivity for cellular clumps, saturation, and inhibition. These features suggest specific binding.

  7. Differentiation of human amniotic epithelial cells into Schwann‑like cells via indirect co‑culture with Schwann cells in vitro.

    PubMed

    Zhu, Shuang; Li, Jiachun; Zhu, Qingtang; Dai, Ting; He, Bo; Zhou, Xiang; Xiang, Jianping; Liu, Xiaolin

    2015-02-01

    Human amniotic epithelial cells (hAECs) exhibit multi‑lineage differentiation ability. The present study investigated the possibility that hAECs possess the potential to differentiate into Schwann‑like cells using an in vitro indirect co‑culture approach. hAECs were isolated via enzymatic digestion, and immunocytochemistry and flow cytometry were performed to identify the hAECs. The hAECs were co‑cultured with Schwann cells (SCs) to differentiate the hAECs into Schwann‑like cells via induced proximity. The expression of typical S‑100 SC markers in the co‑cultured hAECs was determined via immunocytochemistry. For the functional experiments, reverse transcription quantitative polymerase chain reaction (RT‑qPCR) was performed to measure the expression levels of nerve growth factor (NGF), brain‑derived neurotrophic factor (BDNF) and glial cell‑derived neurotrophic factor (GDNF) mRNA. In addition, neurite outgrowth was measured in PC12 cells following co‑culture with the differentiated hAECs. Subsequent to co‑culture with SCs for 21 days, the hAECs exhibited spindle‑like morphology. The immunocytochemistry results revealed that the co‑cultured hAECs expressed S‑100, indicating differentiation into Schwann‑like cells. RT‑qPCR revealed that NGF, BDNF and GDNF expression was upregulated upon differentiation. The average axon length of the PC12 cells increased from 21.32±5.45 to 51.32±8.56 µm subsequent to co‑culture with the differentiated hAECs. These results demonstrate that this indirect co‑culture microenvironment induced the hAECs to differentiate into Schwann‑like cells that exhibited the morphological, phenotypic and functional characteristics of SCs. Therefore, the use of differentiated hAECs that exhibit the characteristics of SCs provides a promising alternative to the present techniques used for peripheral nerve regeneration.

  8. Stat5a increases lactation of dairy cow mammary gland epithelial cells cultured in vitro.

    PubMed

    Liu, Xiao Fei; Li, Meng; Li, Qing Zhang; Lu, Li Min; Tong, Hui Li; Gao, Xue Jun

    2012-10-01

    Signal transducer and activator of transcription 5a (Stat5a) transduces signals of extracellular cytokines and growth factors to the nucleus of mammary gland epithelial cells and thereby regulates gene transcription during pregnancy, lactation, and weaning. However, its function on the milk production of dairy cows needs further investigation. In this experiment, the effects of Stat5a on lactation ability of dairy cow mammary gland epithelial cells (DCMECs) were analyzed. Eukaryotic expression vector pcDNA3.1+-stat5a-αS1 was constructed by inserting stat5a gene into the plasmid vector pcDNA3.1+ and replacing CMV promoter with α-S1-casein 5' flanking sequence. The recombinant vector was stably transfected into DCMECs after geneticin (G418) selection. The proliferation and viability of DCMECs, expression of β-casein and stat5a gene, and the content of lactose were detected. The results showed that stat5a gene in eukaryotic expression vector pcDNA3.1+-stat5a-αS1 was highly expressed in DCMECs and could increase the lactation ability of DCMECs. The associativity of Stat5a with nutrients on the lactation ability of DCMECs was also evaluated. Lysine (Lys), methionine (Met), sodium acetate, β-sodium hydroxybutyrate, and glucose all had more positive effects on the lactation function of DCMECs after pcDNA3.1+-stat5a-αS1 transfection. The proliferation and viability of DCMECs, expression of β-casein and stat5a gene, and contents of lactose and triglyceride were detected. The results revealed that nutrients could promote expression of Stat5a gene to increase lactation of DCMECs. These data help to clarify the function of stat5 gene on lactation and gene regulatory networks linking stat5a.

  9. Eosinophils promote epithelial to mesenchymal transition of bronchial epithelial cells.

    PubMed

    Yasukawa, Atsushi; Hosoki, Koa; Toda, Masaaki; Miyake, Yasushi; Matsushima, Yuki; Matsumoto, Takahiro; Boveda-Ruiz, Daniel; Gil-Bernabe, Paloma; Nagao, Mizuho; Sugimoto, Mayumi; Hiraguchi, Yukiko; Tokuda, Reiko; Naito, Masahiro; Takagi, Takehiro; D'Alessandro-Gabazza, Corina N; Suga, Shigeru; Kobayashi, Tetsu; Fujisawa, Takao; Taguchi, Osamu; Gabazza, Esteban C

    2013-01-01

    Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

  10. Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

    PubMed Central

    Pilgrim, Matthew G.; Lengyel, Imre; Lanzirotti, Antonio; Newville, Matt; Fearn, Sarah; Emri, Eszter; Knowles, Jonathan C.; Messinger, Jeffrey D.; Read, Russell W.; Guidry, Clyde; Curcio, Christine A.

    2017-01-01

    Purpose Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss. PMID:28146236

  11. TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

    PubMed Central

    Schwingshackl, Andreas; Roan, Esra; Teng, Bin; Waters, Christopher M.

    2015-01-01

    Background TREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements. Methods We determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA. Results Cytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells. Conclusion Although cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the

  12. A novel sorbitol transport mechanism in cultured renal papillary epithelial cells

    SciTech Connect

    Siebens, A.W.; Spring, K.R. )

    1989-12-01

    The renal papillary epithelial cell line, GRB-PAP1, accumulates sorbitol when grown in a hypertonic (500 mosmol/kgH2O) bathing medium. When the cells are returned to a 300 mosmol/kgH2O medium, they lose their sorbitol rapidly to the bath. Sorbitol movement across the membranes of these cells was investigated by studying the uptake of radioactive sorbitol and related compounds. Sorbitol uptake increased 71-fold when cells grown in 500 mosmol/kgH2O medium were exposed to a 300 mosmol/kgH2O test solution. The magnitude of the permeability increase was proportional to the size of the change in the osmolality of the bathing medium and not the absolute osmolality. Sorbitol uptake was a linear function of medium sorbitol concentration with no sign of saturation at sorbitol concentrations up to 315 mM. Although the permeability of other polyols was increased when the osmolality was reduced, competition between sorbitol and related sugars and polyols could not be demonstrated. Both the increased sorbitol uptake after a decrease in medium osmolality and the decrease to control permeability after return to the original osmolality were complete within 30 s. A wide variety of transport inhibitors and ion substitutions failed to alter the magnitude of the sorbitol permeability increase. The most effective inhibitor was quinidine, 1 mM reducing sorbitol uptake by 73%. The sorbitol permeability increase could also be blocked by reducing the temperature to 0 degrees C. Nonspecific uptake of sorbitol, such as endocytosis, was shown to be of only minor significance. The large increase in sorbitol permeability and subsequent sorbitol efflux enables these cells to withstand large decreases in osmolality without excessive swelling and consequent damage. A similar compensatory mechanism may operate in vivo in the renal papilla during the onset of diuresis.

  13. Effects of increasing numbers of phagocytic inclusions on human retinal pigment epithelial cells in culture: a model for aging.

    PubMed Central

    Boulton, M; Marshall, J

    1986-01-01

    Cultures of human retinal pigment epithelial cells have been challenged with a number of biological (lipofuscin, melanin, and rod outer segments) and non-biological (latex microspheres) particles at a variety of concentrations. The particles were chosen to include examples of both degradable and non-degradable systems. A range of morphological changes were observed by phase contrast microscopy, and these became more atypical with increasing concentration. At the highest concentration cells had ingested so many particles that many had died and others had ruptured. The time course of these changes indicated a relationship between cellular lytic activity and the capacity of the particle to degrade. The potential of this system as a model for studying senescence is discussed. Images PMID:3790481

  14. Characterization of the dopamine transporter gene expression and binding sites in cultured human amniotic epithelial cells.

    PubMed

    Elwan, Mohamed A; Ishii, Takashi; Sakuragawa, Norio

    2003-05-15

    In this study we sought to investigate whether the dopamine transporter, DAT, and its binding sites are expressed in the human amniotic epithelial cells (HAEC) using reverse transcription-polymerase chain reaction (RT-PCR) and radioligand binding studies, respectively. The RT-PCR findings showed that HAEC expressed DAT mRNA with 100% homology to the human brain DAT. Saturation binding studies using [3H]mazindol showed a high affinity DAT binding site with K(D) and B(max) values of 12.32+/-1.67 nM and 82.7+/-9.74 fmol/mg protein, respectively. Competition experiments showed that selective DAT blockers are potent displacers of [3H]mazindol binding. The rank order of potency of the competing drugs is consistent with the pharmacology of the DAT. The present results provide compelling evidence that HAEC natively express the DAT mRNA and binding sites. More importantly, these results may suggest that HAEC is an appropriate human cell model for studying dopamine release and uptake processes and potential ligands at these sites.

  15. Geldanamycin and its analog induce cytotoxicity in cultured human retinal pigment epithelial cells.

    PubMed

    Wu, Wen-Chuan; Wu, Meng-Hsien; Chang, Yo-Chen; Hsieh, Ming-Chu; Wu, Horng-Jiun; Cheng, Kai-Chun; Lai, Yu-Hung; Kao, Ying-Hsien

    2010-08-01

    Geldanamycin (GA), a benzoquinone ansamycin, was originally isolated as a natural product with anti-fungal activity. GA and its analogs, including 17-allylamino-demethoxy geldanamycin (17-AAG), are also known to block the function of a molecular chaperone, heat shock protein 90 (Hsp90). In light of their anti-tumor properties through direct cytotoxicity and anti-angiogenicity, GA has been previously demonstrated to suppress hypoxia-induced VEGF production in retinal pigment epithelium (RPE) cells, implicating its applicability in treating intraocular neovascularization. This study aimed at investigating the effectiveness of Hsp90 inhibitor treatment in suppressing proliferation of cultured human RPE cells and elucidating its underlying mechanism. Cultured RPE cells were treated with GA or 17-AAG and subjected for cell proliferation assay and cell cycle analysis. Expression of apoptotic regulators and survival signaling activity were monitored by Western blotting. The results showed that both GA and 17-AAG significantly inhibited RPE cell proliferation at micromolar levels. Treatment with GA and 17-AAG led to growth arrests in G1 and S phases, increased sub-G1 hypodipoid cell population, induced apoptotic cell death, and upregulated P53 and P21 expression, although the drug-induced Bcl-2 upregulation cannot prevent cell death. Additionally, GA and 17-AAG significantly suppressed constitutive contents of phosphorylated ERK1/2 and total Akt proteins, and completely abrogated wortmannin-sensitized Akt phosphorylation. In conclusion, GA and 17-AAG inhibit RPE cell proliferation and induce cytotoxicity, possibly through downregulating Akt- and ERK1/2-mediated signaling activities. They might potentially constitute a therapeutic agent for ocular disorders with RPE over proliferation, such as proliferative vitreoretinopathy.

  16. Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells.

    PubMed

    Wilmer, Martijn J; Kluijtmans, Leo A J; van der Velden, Thea J; Willems, Peter H; Scheffer, Peter G; Masereeuw, Rosalinde; Monnens, Leo A; van den Heuvel, Lambertus P; Levtchenko, Elena N

    2011-06-01

    Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.

  17. Monolayer culture systems with respiratory epithelial cells for evaluation of bacterial invasiveness.

    PubMed

    Hirakata, Yoichi; Yano, Hisakazu; Arai, Kazuaki; Endo, Shiro; Kanamori, Hajime; Aoyagi, Tetsuji; Hirotani, Ayako; Kitagawa, Miho; Hatta, Masumitsu; Yamamoto, Natsuo; Kunishima, Hiroyuki; Kawakami, Kazuyoshi; Kaku, Mitsuo

    2010-01-01

    Pseudomonas (P.) aeruginosa is a major opportunistic pathogen especially in immunocompromised patients. To evaluate the invasiveness of respiratory pathogens, we developed monolayer culture systems and examined the degree of invasion by P. aeruginosa and invasive Salmonella (S.) typhimurium strains using human respiratory cell lines: A549 (derived from lung cancer), BEAS-2B (normal bronchial epithelium), and Calu-3 (pleural effusion of a patient with adenocarcinoma of the lung). Cells were seeded into filter units containing 0.33 cm(2) filter membranes with 3.0 microm pores, and were incubated at 37 degrees C under 5% CO(2) for 4-10 days. By monitoring the trans-monolayer electrical resistance (TER), we judged that BEAS-2B cells (TER values: 436.2 +/- 16.8 to 628.8 +/- 66.3 Omega cm(2)) and Calu-3 cells (TER values: 490.5 +/- 25.2 to 547.8 +/- 21.6 Omega cm(2)) formed monolayers with tight junctions, but not A549 cells. On day 8 of culture, monolayer cultures were infected with bacteria, and the number of microorganisms penetrating into the basolateral medium was counted. Wild-type P. aeruginosa PAO1 (PAO1 WT) and S. typhimurium SL1344 were detected in the basolateral medium of BEAS-2B monolayer system by 3 h after inoculation, while only P. aeruginosa PAO1 WT was detected in the basolateral medium of Calu-3 monolayer, indicating poor invasiveness of S. typhimurium SL1344 in the Calu-3 system. These findings suggest that BEAS-2B or Calu-3 monolayer system could be useful for evaluating the invasiveness of respiratory pathogens. Because of the difference in bacterial invasiveness, we may need to choose a suitable cell system for each target pathogen.

  18. Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture.

    PubMed Central

    Takahashi, M; Ota, S; Shimada, T; Hamada, E; Kawabe, T; Okudaira, T; Matsumura, M; Kaneko, N; Terano, A; Nakamura, T

    1995-01-01

    Various growth factors are suggested to be involved in gastric mucosal repair. Our previous studies have shown that exogenous hepatocyte growth factor (HGF) has a proliferative effect on gastric epithelial cells. In the present study, comparison of the maximum proliferative effects and the optimum concentrations of several growth factors revealed that HGF was the most potent mitogen for gastric epithelial cells, as is the case for hepatocytes. Restitution of gastric epithelial cell monolayers was assessed using a round wound restitution model. HGF was the most effective agent for facilitating gastric epithelial restitution among those tested. A binding assay revealed specific binding of HGF to its receptor on gastric epithelial cells. Northern blot analysis confirmed the expression of specific HGF receptor mRNA (c-met) by gastric epithelial cells but not by gastric fibroblasts. To investigate endogenous HGF production, we determined the effect of gastric fibroblast-conditioned medium on epithelial proliferation and restitution. The conditioned medium produced similar effects to HGF and its activity was neutralized by an anti-HGF antibody. In addition, expression of HGF mRNA was detected in gastric fibroblasts but not in gastric epithelial cells. Our immunohistochemical study confirmed these in vitro data by means of demonstrating the existence and localization of HGF at human native gastric mucosa. HGF was localized at fibroblasts under the epithelial cell layer around gastric ulcers. These results suggest that HGF may be a potent endogenous promotor of gastric epithelial cell proliferation and migration, and may contribute to gastric mucosal repair through a paracrine mechanism. Images PMID:7738166

  19. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells.

    PubMed

    Economou, E C; Marinelli, S; Smith, M C; Routt, A A; Kravets, V V; Chu, H W; Spendier, K; Celinski, Z J

    2016-09-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1-100 nm in diameter were prepared in water. BaNPs and conventional 20-30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles.

  20. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells

    PubMed Central

    Economou, E. C.; Marinelli, S.; Smith, M. C.; Routt, A. A.; Kravets, V. V.; Chu, H. W.; Spendier, K.; Celinski, Z. J.

    2016-01-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1–100 nm in diameter were prepared in water. BaNPs and conventional 20–30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles. PMID:27774374

  1. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

    PubMed

    Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

    2014-12-01

    Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors.

  2. Autosomal mutations in mouse kidney epithelial cells exposed to high-energy protons in vivo or in culture.

    PubMed

    Turker, Mitchell S; Grygoryev, Dmytro; Dan, Cristian; Eckelmann, Bradley; Lasarev, Michael; Gauny, Stacey; Kwoh, Ely; Kronenberg, Amy

    2013-05-01

    Proton exposure induces mutations and cancer, which are presumably linked. Because protons are abundant in the space environment and significant uncertainties exist for the effects of space travel on human health, the purpose of this study was to identify the types of mutations induced by exposure of mammalian cells to 4-5 Gy of 1 GeV protons. We used an assay that selects for mutations affecting the chromosome 8-encoded Aprt locus in mouse kidney cells and selected mutants after proton exposure both in vivo and in cell culture. A loss of heterozygosity (LOH) assay for DNA preparations from the in vivo-derived kidney mutants revealed that protons readily induced large mutational events. Fluorescent in situ hybridization painting for chromosome 8 showed that >70% of proton-induced LOH patterns resembling mitotic recombination were in fact the result of nonreciprocal chromosome translocations, thereby demonstrating an important role for DNA double-strand breaks in proton mutagenesis. Large interstitial deletions, which also require the formation and resolution of double-strand breaks, were significantly induced in the cell culture environment (14% of all mutants), but to a lesser extend in vivo (2% of all mutants) suggesting that the resolution of proton-induced double-strand breaks can differ between the intact tissue and cell culture microenvironments. In total, the results demonstrate that double-strand break formation is a primary determinant for proton mutagenesis in epithelial cell types and suggest that resultant LOH for significant genomic regions play a critical role in proton-induced cancers.

  3. Control of lens epithelial cell survival

    PubMed Central

    1993-01-01

    We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell- cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture. PMID:8491781

  4. Cementum- and periodontal ligament-like tissue formation by dental follicle cell sheets co-cultured with Hertwig's epithelial root sheath cells.

    PubMed

    Bai, Yudi; Bai, Yuxiang; Matsuzaka, Kenichi; Hashimoto, Sadamitsu; Fukuyama, Tatsuro; Wu, Lian; Miwa, Tsuneyuki; Liu, Xiaohui; Wang, Xiaojing; Inoue, Takashi

    2011-06-01

    Dental follicle cells (DFCs) are believed contain the precursor cells of the periodontium and can form cell sheets by secreting extracellular matrix (ECM) proteins. Cell sheet engineering has been recently developed and applied successfully in the field of tissue regeneration. However, research on the in vitro characteristics of DFC sheets is lacking and an assessment of whether DFC sheets can produce periodontal tissues in vivo has not been reported. To test the characteristics and applicability of DFC sheets in this field, we established a co-culture system of rat DFCs and Hertwig's epithelial root sheath (HERS) cells in vitro, and included the following controls: a co-culture of DFCs and alveolar mucosa epithelial cells, DFCs with no cells in the upper chamber, and DFCs cultured without an upper chamber. After 3 weeks of co-culturing the cells, the DFC sheets were transplanted into adult male rats' omenta. One week after co-culturing DFCs with HERS cells, mRNA levels of collagen type I (COL-1), alkaline phosphatase (ALP), runt related transcription factor 2 (Runx 2) and bone sialoprotein (BSP) were increased significantly. In addition, after 3 weeks of co-culturing the cells, the numbers of ALP-, osteocalcin (OCN)-, BSP- and osteoprotegerin (OPG)-positive DFCs increased. The DFCs also produced more calcified nodules and exhibited an increased number of subcellular organelles, which are important for protein synthesis and secretion. Moreover, gap junctions were found between the experimental DFCs within the sheet. Five weeks of in vivo growth of DFC sheets pre-exposed to HERS cells led to the formation of cementum-like tissues, which were positive for OCN, BSP and OPG, as well as the formation of periodontal ligament-like tissues, which were positive for COL-1. In contrast, control cells only produced fibrous tissues. These results indicate that the DFC sheets induced by HERS cells are able to produce periodontal tissues through epithelial

  5. Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures

    PubMed Central

    Mrozowska, Paulina S.

    2016-01-01

    MDCK II cells, a widely used model of polarized epithelia, develop into different structures depending on culture conditions: two-dimensional (2D) monolayers when grown on synthetic supports or three-dimensional (3D) cysts when surrounded by an extracellular matrix. The establishment of epithelial polarity is accompanied by transcytosis of the apical marker podocalyxin from the outer plasma membrane to the newly formed apical domain, but its exact route and regulation remain poorly understood. Here, through comprehensive colocalization and knockdown screenings, we identified the Rab GTPases mediating podocalyxin transcytosis and showed that different sets of Rabs coordinate its transport during cell polarization in 2D and 3D structures. Moreover, we demonstrated that different Rab35 effectors regulate podocalyxin trafficking in 2D and 3D environments; trafficking is mediated by OCRL in 2D monolayers and ACAP2 in 3D cysts. Our results give substantial insight into regulation of the transcytosis of this apical marker and highlight differences between trafficking mechanisms in 2D and 3D cell cultures. PMID:27138252

  6. Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model

    PubMed Central

    Gagliano, Nicoletta; Celesti, Giuseppe; Tacchini, Lorenza; Pluchino, Stefano; Sforza, Chiarella; Rasile, Marco; Valerio, Vincenza; Laghi, Luigi; Conte, Vincenzo; Procacci, Patrizia

    2016-01-01

    AIM: To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. METHODS: HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, β-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS: The E-cadherin/β-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/β-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression. CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids. PMID:27182158

  7. Effects of shiga toxin 2 on cellular regeneration mechanisms in primary and three-dimensional cultures of human renal tubular epithelial cells.

    PubMed

    Márquez, Laura B; Araoz, Alicia; Repetto, Horacio A; Ibarra, Fernando R; Silberstein, Claudia

    2016-10-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) causes post-diarrheal Hemolytic Uremic Syndrome (HUS), which is one of the most common causes of acute renal failure in children in Argentine. The aim of the present work was to study the effects of Shiga toxin type 2 (Stx2) on regenerative mechanisms of primary cultures of human cortical renal tubular epithelial cells (HRTEC) and three-dimensional (3D) cultures of HRTEC. Primary cultures of HRTEC were able to develop tubular structures when grown in matrigel, which showed epithelial cells surrounding a central lumen resembling the original renal tubules. Exposure to Stx2 inhibited tubulogenesis in 3D-HRTEC cultures. Moreover, a significant increase in apoptosis, and decrease in cell proliferation was observed in tubular structures of 3D-HRTEC exposed to Stx2. A significant reduction in cell migration and vimentin expression levels was observed in HRTEC primary cultures exposed to Stx2, demonstrating that the holotoxin affected HRTEC dedifferentiation. Furthermore, a decreased number of cells expressing CD133 progenitor marker was found in HRTEC cultures treated with Stx2. The CD133 positive cells also expressed the Stx receptor globotriaosylceramide, which may explain their sensitivity to Stx2. In conclusion, Stx2 affects the regenerative processes of human renal tubular epithelial cells in vitro, by inhibiting cell dedifferentiation mechanisms, as well as tubules restoration. The development of 3D-HRTEC cultures that resemble original human renal proximal tubules is a novel in vitro model to study renal epithelial repair mechanisms after injury.

  8. Direct binding of boar ejaculate and epididymal spermatozoa to porcine epididymal epithelial cells is also needed to maintain sperm survival in in vitro co-culture.

    PubMed

    Yeste, Marc; Castillo-Martín, Míriam; Bonet, Sergi; Briz, Maria Dolors

    2012-04-01

    The aim of the present study was to compare the influence of cultured epididymal epithelial cells (EEC) from corpus, caput or cauda, oviductal epithelial cells (OEC) and non-reproductive epithelial cells (LLC-PK1) on function and survival of epididymal and ejaculated spermatozoa, in the latter case to determine whether such influence differed between morphologically normal and abnormal spermatozoa. For this purpose, either spermatozoa were directly co-cultured with EEC from caput, corpus, or cauda, OEC and LLC-PK1 cells (experiment 1) or a membrane-diffusible insert was included in these co-cultures (experiment 2). EEC cultured from the three epididymal regions did not differently affect the sperm parameters. Morphologically normal spermatozoa presented a higher ability to bind EEC, OEC, and LLC-PK1 than abnormal spermatozoa with cytoplasmic droplets or with tail/head malformations. Epididymal spermatozoa were more able to bind EEC during the first 24 h of co-culture, while ejaculated spermatozoa presented a higher capacity to bind OEC between 30 min and 3 h of co-incubation. In all cases, the ability to bind to epithelial cells was higher when they were co-cultured with EEC and OEC than with LLC-PK1. After 2 h of co-culture, the viability of epididymal spermatozoa was better maintained when they bound EEC than when they bound OEC. Conversely, the viability of ejaculated spermatozoa was better maintained when bound OEC than when bound EEC after 24 and 48 h of co-culture. Our work, apart from corroborating the involvement of morphologically normal spermatozoa in the formation of sperm reservoir, highlights the importance of direct contact spermatozoa-EEC in maintaining the sperm survival in in vitro co-culture, and also suggests that a specific binding between EEC and epididymal spermatozoa exists.

  9. Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. II. Cytokine activities in murine thymic epithelial and mesenchymal cell culture supernatants.

    PubMed

    Eshel, I; Savion, N; Shoham, J

    1990-03-01

    Two morphologically distinct primary cultures of murine thymic stroma were established and found to be of epithelial (MTEC) and mesenchymal (MTMC) origin. These cultures were generated by selective conditions of tissue disruption and were maintained on extracellular matrix in defined medium. Culture supernatants (CS) from these cultures (EC-CS and MC-CS respectively), were tested for cytokine production and for effects on thymocyte maturation. Both supernatants displayed the activities of IL-3 and of granulocyte/macrophage-CSF and not of IL-1, -2, -4, or IFN. In addition they were found to be mitogenic to murine thymocytes in a "spontaneous" [3H]TdR incorporation assay. The two supernatants differed, however, in their effect on Con A stimulation. EC-CS had a strong enhancing effect, both when used for preincubation (18 h) before Con A stimulation or when present simultaneously with it. MC-CS had a small inconsistent effect under these conditions. Also EC-CS enhanced IL-2 and IL-3 production by thymocytes. The responsive thymocyte subpopulation was the one that does not bind peanut agglutinin. CS of an established thymic epithelial cell line displayed only part of these activities at a considerably lower level. CS from primary kidney cell culture was completely devoid of activity. The results suggest that primary thymic stromal cell cultures, cultivated under the defined conditions described here, may better preserve physiologic secretory activities, and probably also other cell functions, compared with established cell lines. Furthermore, the results are compatible with the hypothesis that the soluble factors, secreted by thymic stromal cells, are active on either very early or late stages of thymic differentiation, whereas the main intrathymic stages of differentiation are conceivable dependent primarily on direct contact with stromal cells.

  10. Bovine Oviduct Epithelial Cells Dedifferentiate Partly in Culture, While Maintaining their Ability to Improve Early Embryo Development Rate and Quality.

    PubMed

    Schmaltz-Panneau, B; Locatelli, Y; Uzbekova, S; Perreau, C; Mermillod, P

    2015-10-01

    There are convincing arguments to suggest that the success of early reproductive events is reliant on a satisfactory dialogue between gametes-embryo and the oviduct epithelium. The aim of this study was to develop and characterize an in vitro model to study these interactions. Cattle zygotes produced in vitro were cultured in either SOF or TCM-199 in the presence or absence of bovine oviduct cell monolayers (BOEC), under 20% or 5% O2 . The embryonic development rate and its quality (cell numbers, cryosurvival) were evaluated, as were the BOEC contents in 11 candidate transcripts (real-time PCR) at different time points. A BOEC co-culture did indeed increase the rate of development in both media under 5% O2 (41 vs 27% and 28 vs 10% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). The effect of BOEC on the developmental rate was more pronounced under 20% O2 (35 vs 6% and 27 vs 4% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). BOEC significantly increased the embryonic cell count in TCM-199 (122.5 ± 11.1 vs 70.3 ± 9.6; p < 0.05) and embryonic cryosurvival in both media. The expression levels of SOD, FGF2 and TGF-β1 in BOEC remained steady during culture, although mRNA levels of OGP, C3, PGR and ESR2 were clearly reduced, suggesting a dedifferentiation of BOEC during culture. However, SSP1 and GPX4 transcripts were slightly increased during culture, this rise becoming significant by the end of the culture period. In conclusion, our co-culture system with bovine oviduct epithelial cells used for the development of bovine zygotes produced in vitro enhanced blastocyst formation and above all the quality of the resulting embryos, which was associated with specific transcriptomic changes.

  11. Precision-cut intestinal slices as a culture system to analyze the infection of differentiated intestinal epithelial cells by avian influenza viruses.

    PubMed

    Punyadarsaniya, Darsaniya; Winter, Christine; Mork, Ann-Kathrin; Amiri, Mahdi; Naim, Hassan Y; Rautenschlein, Silke; Herrler, Georg

    2015-02-01

    Many viruses infect and replicate in their host via the intestinal tract, e.g. many picornaviruses, several coronaviruses and avian influenza viruses of waterfowl. To analyze infection of enterocytes is a challenging task as culture systems for differentiated intestinal epithelial cells are not readily available and often have a life span that is too short for infection studies. Precision-cut intestinal slices (PCIS) from chicken embryos were prepared and shown that the epithelial cells lining the lumen of the intestine are viable for up to 4 days. Using lectin staining, it was demonstrated that α2,3-linked sialic acids, the preferred receptor determinants of avian influenza viruses, are present on the apical side of the epithelial cells. Furthermore, the epithelial cells (at the tips) of the villi were shown to be susceptible to infection by an avian influenza virus of the H9N2 subtype. This culture system will be useful to analyze virus infection of intestinal epithelial cells and it should be applicable also to the intestine of other species.

  12. Interphase Chromosome Conformation and Chromatin-Chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

  13. Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

    PubMed Central

    Szober, Christoph M.; Hauck, Stefanie M.; Euler, Kerstin N.; Fröhlich, Kristina J. H.; Alge-Priglinger, Claudia; Ueffing, Marius; Deeg, Cornelia A.

    2012-01-01

    The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies. PMID:23203049

  14. Isospora suis in an Epithelial Cell Culture System – An In Vitro Model for Sexual Development in Coccidia

    PubMed Central

    Worliczek, Hanna Lucia; Ruttkowski, Bärbel; Schwarz, Lukas; Witter, Kirsti; Tschulenk, Waltraud; Joachim, Anja

    2013-01-01

    Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isosporasuis, a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I. suis, including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (≤1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I. suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia. PMID:23861983

  15. High-frequency low-level diode laser irradiation promotes proliferation and migration of primary cultured human gingival epithelial cells.

    PubMed

    Ejiri, Kenichiro; Aoki, Akira; Yamaguchi, Yoko; Ohshima, Mitsuhiro; Izumi, Yuichi

    2014-07-01

    In periodontal therapy, the use of low-level diode lasers has recently been considered to improve wound healing of the gingival tissue. However, its effects on human gingival epithelial cells (HGECs) remain unknown. The aim of the present study was to examine whether high-frequency low-level diode laser irradiation stimulates key cell responses in wound healing, proliferation and migration, in primary cultured HGECs in vitro. HGECs were derived from seven independent gingival tissue specimens. Cultured HGECs were exposed to a single session of high-frequency (30 kHz) low-level diode laser irradiation with various irradiation time periods (fluence 5.7-56.7 J/cm(2)). After 20-24 h, cell proliferation was evaluated by WST-8 assay and [(3)H]thymidine incorporation assay, and cell migration was monitored by in vitro wound healing assay. Further, phosphorylation of the mitogen-activated protein kinase (MAPK) pathways after irradiation was investigated by Western blotting. The high-frequency low-level irradiation significantly increased cell proliferation and [(3)H]thymidine incorporation at various irradiation time periods. Migration of the irradiated cells was significantly accelerated compared with the nonirradiated control. Further, the low-level diode laser irradiation induced phosphorylation of MAPK/extracellular signal-regulated protein kinase (ERK) at 5, 15, 60, and 120 min after irradiation. Stress-activated protein kinases/c-Jun N-terminal kinase and p38 MAPK remained un-phosphorylated. The results show that high-frequency low-level diode laser irradiation promotes HGEC proliferation and migration in association with the activation of MAPK/ERK, suggesting that laser irradiation may accelerate gingival wound healing.

  16. Isospora suis in an epithelial cell culture system - an in vitro model for sexual development in coccidia.

    PubMed

    Worliczek, Hanna Lucia; Ruttkowski, Bärbel; Schwarz, Lukas; Witter, Kirsti; Tschulenk, Waltraud; Joachim, Anja

    2013-01-01

    Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isosporasuis, a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I. suis, including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (≤1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I. suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia.

  17. Isolation, culture and characterization of biliary epithelial cells from different anatomical levels of the intrahepatic and extrahepatic biliary tree from a mouse.

    PubMed

    Katayanagi, K; Kono, N; Nakanuma, Y

    1998-04-01

    We developed methods to isolate biliary epithelial cells (BECs) from the gallbladder (GB), common bile duct (CBD), intrahepatic large bile duct (ILBD) and small bile duct (ISBD) of a mouse, simultaneously. ILBD and ISBD were cut from the biliary tree after collagenase perfusion of the liver. BECs from all of these biliary segments were cultured as explants on collagen gel. BECs spread from the explants and formed cellular sheets. Areas of these sheets composed entirely of BECs were cut and placed on other gels as subculture, and this continued for 10 passages. Primary and passage cultured BECs on gel were composed of a monolayer of epithelial cells. Passaged cultured BECs in gel formed a spherical cyst lined by a single epithelial layer. Ultrastructurally, microvilli were dense on the luminal surface, and junctional complex and interdigitation was identifiable on the lateral surfaces. These features were similar in both primary and passaged cultured BECs, irrespective of their anatomical origin. Major histocompatibility complex antigens and intercellular adhesion molecule-1 were induced on the basolateral cell membranes of primary and passaged cultured BECs, by interferon-gamma. Although several phenotypic, structural and probable biological features of BECs inherent to each anatomical level may be lost after culture on gel, a combination of this method, several immunological modifications in experimental animals, and addition of immunologically active substances to the culture medium will make the immunopathologic analysis of biliary diseases possible.

  18. Pro-inflammatory Cytokines Impair Vitamin D-induced Host Defense in Cultured Airway Epithelial Cells.

    PubMed

    Schrumpf, Jasmijn A; Amatngalim, Gimano D; Veldkamp, Joris B; Verhoosel, Renate M; Ninaber, Dennis K; Ordonez, Soledad R; van der Does, Anne M; Haagsman, Henk P; Hiemstra, Pieter S

    2017-02-23

    Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitamin D metabolism and action. We hypothesized that long-term exposure of primary bronchial epithelial cells (PBEC) to pro-inflammatory cytokines alters their vitamin D metabolism, antibacterial activity and expression of hCAP18/LL-37. To investigate this, PBEC were differentiated at the air-liquid interphase for 14 days in presence of the pro-inflammatory cytokines TNF-α and IL-1β (TNF-α/IL-1β), and subsequently exposed to vitamin D (inactive 25(OH)D3 and active 1,25(OH)2D3). Expression of hCAP18/LL-37, vitamin D receptor (VDR) and enzymes involved in vitamin D metabolism (CYP24A1 and CYP27B1) was determined using qPCR, Western blot and immunofluorescence staining. Furthermore, vitamin D-mediated antibacterial activity was assessed using non-typeable Haemophilus influenzae (NTHi). We found that TNF-α/IL-1β treatment reduced vitamin D-induced expression of hCAP18/LL-37 and killing of NTHi. In addition, CYP24A1 (a vitamin D-degrading enzyme) was increased by TNF-α/IL-1β, whereas CYP27B1 (that converts 25(OH)D3 to its active form) and VDR expression remained unaffected. Furthermore, we demonstrated that the TNF-α/IL-1β-mediated induction of CYP24A1 was at least in part mediated by the transcription factor specific protein 1 (Sp1) and the EGFR-MAPK-pathway. These findings indicate that TNF-α/IL-1β decreases vitamin D-mediated antibacterial activity and hCAP18/LL-37 expression via induction of CYP24A1, and suggests that chronic inflammation impairs protective responses induced by vitamin D.

  19. Plasma polymer-coated contact lenses for the culture and transfer of corneal epithelial cells in the treatment of limbal stem cell deficiency.

    PubMed

    Brown, Karl David; Low, Suet; Mariappan, Indumathi; Abberton, Keren Maree; Short, Robert; Zhang, Hong; Maddileti, Savitri; Sangwan, Virender; Steele, David; Daniell, Mark

    2014-02-01

    Extensive damage to the limbal region of the cornea leads to a severe form of corneal blindness termed as limbal stem cell deficiency (LSCD). Whereas most cases of corneal opacity can be treated with full thickness corneal transplants, LSCD requires stem cell transplantation for successful ocular surface reconstruction. Current treatments for LSCD using limbal stem cell transplantation involve the use of murine NIH 3T3 cells and human amniotic membranes as culture substrates, which pose the threat of transmission of animal-derived pathogens and donor tissue-derived cryptic infections. In this study, we aimed to produce surface modified therapeutic contact lenses for the culture and delivery of corneal epithelial cells for the treatment of LSCD. This approach avoids the possibility of suture-related complications and is completely synthetic. We used plasma polymerization to deposit acid functional groups onto the lenses at various concentrations. Each surface was tested for its suitability to promote corneal epithelial cell adhesion, proliferation, retention of stem cells, and differentiation and found that acid-based chemistries promoted better cell adhesion and proliferation. We also found that the lenses coated with a higher percentage of acid functional groups resulted in a higher number of cells transferred onto the corneal wound bed in rabbit models of LSCD. Immunohistochemistry of the recipient cornea confirmed the presence of autologous, transplanted 5-bromo-2'-deoxyuridine (BrdU)-labeled cells. Hematoxylin staining has also revealed the presence of a stratified epithelium at 26 days post-transplantation. This study provides the first evidence for in vivo transfer and survival of cells transplanted from a contact lens to the wounded corneal surface. It also proposes the possibility of using plasma polymer-coated contact lenses with high acid functional groups as substrates for the culture and transfer of limbal cells in the treatment of LSCD.

  20. Plasma Polymer-Coated Contact Lenses for the Culture and Transfer of Corneal Epithelial Cells in the Treatment of Limbal Stem Cell Deficiency

    PubMed Central

    Brown, Karl David; Low, Suet; Mariappan, Indumathi; Abberton, Keren Maree; Short, Robert; Zhang, Hong; Maddileti, Savitri; Sangwan, Virender; Steele, David

    2014-01-01

    Extensive damage to the limbal region of the cornea leads to a severe form of corneal blindness termed as limbal stem cell deficiency (LSCD). Whereas most cases of corneal opacity can be treated with full thickness corneal transplants, LSCD requires stem cell transplantation for successful ocular surface reconstruction. Current treatments for LSCD using limbal stem cell transplantation involve the use of murine NIH 3T3 cells and human amniotic membranes as culture substrates, which pose the threat of transmission of animal-derived pathogens and donor tissue-derived cryptic infections. In this study, we aimed to produce surface modified therapeutic contact lenses for the culture and delivery of corneal epithelial cells for the treatment of LSCD. This approach avoids the possibility of suture-related complications and is completely synthetic. We used plasma polymerization to deposit acid functional groups onto the lenses at various concentrations. Each surface was tested for its suitability to promote corneal epithelial cell adhesion, proliferation, retention of stem cells, and differentiation and found that acid-based chemistries promoted better cell adhesion and proliferation. We also found that the lenses coated with a higher percentage of acid functional groups resulted in a higher number of cells transferred onto the corneal wound bed in rabbit models of LSCD. Immunohistochemistry of the recipient cornea confirmed the presence of autologous, transplanted 5-bromo-2′-deoxyuridine (BrdU)-labeled cells. Hematoxylin staining has also revealed the presence of a stratified epithelium at 26 days post-transplantation. This study provides the first evidence for in vivo transfer and survival of cells transplanted from a contact lens to the wounded corneal surface. It also proposes the possibility of using plasma polymer-coated contact lenses with high acid functional groups as substrates for the culture and transfer of limbal cells in the treatment of LSCD. PMID:24328453

  1. Culture of Primary Ciliary Dyskinesia Epithelial Cells at Air-Liquid Interface Can Alter Ciliary Phenotype but Remains a Robust and Informative Diagnostic Aid

    PubMed Central

    Coles, Janice L.; Williams, Gwyneth; Rutman, Andrew; Goggin, Patricia M.; Adam, Elizabeth C.; Page, Anthony; Evans, Hazel J.; Lackie, Peter M.; O’Callaghan, Christopher; Lucas, Jane S.

    2014-01-01

    Background The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns. Methods We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n = 111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture. Results Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced. Conclusions The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia. PMID:24586956

  2. Patterning Bacterial Communities on Epithelial Cells

    PubMed Central

    Dwidar, Mohammed; Leung, Brendan M.; Yaguchi, Toshiyuki; Takayama, Shuichi; Mitchell, Robert J.

    2013-01-01

    Micropatterning of bacteria using aqueous two phase system (ATPS) enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv) gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions. PMID:23785519

  3. Qualitatively monitoring binding and expression of the transcription factor Sp1 as a useful tool to evaluate the reliability of primary cultured epithelial stem cells in tissue reconstruction.

    PubMed

    Gaudreault, Manon; Larouche, Danielle; Germain, Lucie; Guérin, Sylvain L

    2013-01-01

    Electrophoretic mobility shift assay and Western blot are simple, efficient, and rapid methods for the study of DNA-protein interactions and expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem-cells through the culture process is essential to produce high quality substitutes. However as such cells are passaged in culture, they often lose their ability to proliferate, a process likely to be determined by the altered expression of nuclear-located transcription factors such as Sp1, whose expression has been documented to be required for cell adhesion, migration, and differentiation. Our recent studies demonstrated that reconstructed tissues exhibiting poor histological and structural characteristics are also those that were produced with epithelial cells in which expression and DNA binding of Sp1 was reduced in vitro. Therefore, monitoring both the expression and DNA binding of this transcription factor in human skin and corneal epithelial cells might prove a particularly useful tool for selecting which cells are to be used for tissue reconstruction.

  4. A 3-D airway epithelial cell and macrophage co-culture system to study Rhodococcus equi infection.

    PubMed

    Schwab, Ute; Caldwell, Shannon; Matychak, Mary-Beth; Felippe, Julia

    2013-07-15

    We developed a 3-D equine bronchial epithelial cell (BEC) culture that fully differentiates into ciliary beating and mucus producing cells. Using this system, we evaluated how mucus affects the phagocytic activity of macrophages. Adult horse monocyte-derived macrophages were incubated with Rhodococcus equi for 4h either in the mucus layer of in vitro generated airway epithelium or on collagen coated membranes. Using light and electron microscopy, we noted that the number of macrophages with intracellular bacteria, and the number of intracellular bacteria per macrophage were lower in the presence of mucus. TNFα measurements revealed that the presence of BECs promoted TNFα production by R. equi-infected macrophages; a decrease in TLR-2 (involved in R. equi recognition) and an increase in EGF-R (involved in mucin production) mRNA expression were also noted. Interestingly, when foal macrophages were added to foal BECs, we made the opposite observation, i.e. many macrophages were loaded with R. equi. Our in vitro bronchial system shows great potential for the identification of mechanisms how BECs and mucus play a role in phagocyte activation and bacterial clearance. Further studies using this system will show whether the airway environment in the foal responds differently to R. equi infection.

  5. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

    PubMed

    Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

    2016-03-04

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

  6. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    PubMed Central

    Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  7. Effects of cell culture techniques on gene expression and cholesterol efflux in primary bovine mammary epithelial cells derived from milk and tissue.

    PubMed

    Sorg, D; Potzel, A; Beck, M; Meyer, H H D; Viturro, E; Kliem, H

    2012-10-01

    Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p ≤ 0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.

  8. Viable and morphologically normal boar spermatozoa alter the expression of heat-shock protein genes in oviductal epithelial cells during co-culture in vitro.

    PubMed

    Yeste, Marc; Holt, William V; Bonet, Sergi; Rodríguez-Gil, Joan E; Lloyd, Rhiannon E

    2014-09-01

    The principal aim of this study was to determine if boar spermatozoa influence the expression of four selected chaperone and heat-shock protein (HSP) genes-namely clusterin (CLU), HSP90AA1, HSPA5, and HSPA8-in oviductal epithelial cells (OECs) during in vitro co-culture. All corresponding proteins of these genes were previously identified in a sperm-interacting, 70-kDa soluble fraction derived from apical plasma membranes of OECs. The present study also sought to determine whether or not: (i) spermatozoa must directly bind to OEC for an effect on gene expression to be elicited and (ii) reproductive and nonreproductive epithelial cell types (LLC-PK1, pig kidney) respond equivalently, in terms of alterations in chaperone and HSP gene expression, during co-culture with sperm. Spermatozoa induced a significant upregulation (P < 0.05) in HSP90AA1 and HSPA5 in OECs after 3 hr, and in HSPA8 after 6 hr of co-culture when they were in direct contact with epithelial cells. Conversely, no upregulation of HSP transcription was observed when spermatozoa did not directly bind to OECs. Spermatozoa also induced a significant upregulation (P < 0.05) of the same three genes when in direct contact with LLC-PK1 cells, but the timing occurred later than with OECs. Interestingly, the extent of HSP gene upregulation induced by direct contact of spermatozoa with epithelial cells was dependent on sperm-binding index and on the viability and morphological quality of the bound sperm population. In conclusion, the upregulation of HSP genes caused by direct contact between spermatozoa and OECs, rather than nonreproductive epithelial cells, suggests HSPs could play an integral role in the modulation of sperm function in the oviductal reservoir.

  9. Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells - Relevance to Capsular Opacification.

    PubMed

    Recek, Nina; Andjelić, Sofija; Hojnik, Nataša; Filipič, Gregor; Lazović, Saša; Vesel, Alenka; Primc, Gregor; Mozetič, Miran; Hawlina, Marko; Petrovski, Goran; Cvelbar, Uroš

    2016-01-01

    Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO) due to remaining lens epithelial cells (LECs) by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation) at a highly targeted cell level compared to treatment on top of the medium (indirect treatment). Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity) to the posterior lens capsule and PCO formation.

  10. Matrix-based three-dimensional culture of buffalo mammary epithelial cells showed higher induction of genes related to milk protein and fatty acid metabolism.

    PubMed

    Shandilya, Umesh K; Sharma, Ankita; Sodhi, Monika; Kapila, Neha; Kishore, Amit; Mohanty, Ashok; Kataria, Ranjit; Malakar, Dhruva; Mukesh, Manishi

    2016-02-01

    Demanding transcriptomic studies in livestock animal species could be replaced by good in vitro models mimicking the function of mammary gland. Mammary epithelial cells (MEC) are the functional unit of the mammary gland. Extracellular matrix is known to be a key factor providing normal homeostasis in three-dimensional (3D) environment as important signals are lost when cells are cultured in two-dimensional (2D) environment. The aims of this study were to establish a buffalo mammary epithelial cells (BMECs) in 3D culture using extracellular matrix and to determine whether such a 3D culture model has different expression pattern than 2D counterpart. The purified MEC generated after several passages were used to establish 3D culture using Geltrex matrix. The expression of milk casein genes viz., alpha S1-casein (CSN1S1), alpha S2-casein (CSN1S2), beta-casein (CSN2), kappa-casein (CSN3); and fatty acid metabolism genes viz., butyrophilin (BTN1A1), glycerol-3-phosphate acyltransferase (GPAM), fatty acid-binding protein 3 (FABP3), and stearoyl-CoA desaturase (SCD) was assessed in 3D culture in comparison to traditional monolayer culture using qRT-PCR. Notable morphological differences were observed for BMECs grown in 3D culture in comparison to 2D culture. Morphologically, epithelial structures grown in Geltrex matrix (3D) environment showed enhanced functional differentiation in comparison to 2D culture. In 3D culture, lumen and dome-like structures were formed by day 5, whereas polarized acinus-like structure were formed within 15 days of culturing. The expression data showed higher mRNA induction of milk casein and fatty acid metabolism genes in 10-day-old 3D BMECs culture in comparison to 2D monolayer culture. The result suggests that 3D organization of epithelial cells has favorable effect on induction of milk and fatty acid metabolism-related genes. Therefore, matrix-based 3D culture of MEC that recapitulate the structural and functional context of normal tissues

  11. Suppression of interleukin 1α and interleukin 1β in human limbal epithelial cells cultured on the amniotic membrane stromal matrix

    PubMed Central

    Solomon, A.; Rosenblatt, M.; Monroy, D.; Ji, Z.; Pflugfelder, S.; Tseng, S.

    2001-01-01

    AIMS—Amniotic membrane (AM) transplantation reduces inflammation in a variety of ocular surface disorders. The aim of this study was to determine if AM stroma suppresses the expression of the IL-1 gene family in cultured human corneal limbal epithelial cells.
METHODS—Human corneal limbal epithelial cells were cultured from limbocorneal explants of donor eyes on plastic or on the AM stroma. Transcript expression of IL-1α, IL-1β, IL-1 receptor antagonist (RA), and GAPDH was compared with or without addition of lipopolysaccharide to their serum-free media for 24 hours using RNAse protection assay (RPA). Their protein production in the supernatant was analysed by ELISA.
RESULTS—Expression of IL-1α and IL-1β transcripts and proteins was significantly reduced by cells cultured on the AM stromal matrix compared with plastic cultures whether lipopolysaccharide was added or not. Moreover, expression of IL-1 RA by cells cultured in the lipopolysaccharide-free medium was upregulated by AM stromal matrix. The ratio between IL-1 RA and IL-1α protein levels in AM cultures was higher than in plastic cultures.
CONCLUSIONS—AM stromal matrix markedly suppresses lipopolysaccharide induced upregulation of both IL-1α and IL-1β. These data may explain in part the effect of AM transplantation in reducing ocular surface inflammation, underscoring the unique feature of the AM as a substrate for tissue engineering.

 PMID:11264135

  12. Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells.

    PubMed

    Nishi, Hiroyuki; Maeda, Noriko; Izumi, Shunsuke; Higa-Nakamine, Sayomi; Toku, Seikichi; Kakinohana, Manabu; Sugahara, Kazuhiro; Yamamoto, Hideyuki

    2015-02-05

    In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms.

  13. Diesel Exhaust Influences Carcinogenic PAH-Induced Genotoxicity and Gene Expression in Human Breast Epithelial Cells in Culture

    PubMed Central

    Courter, Lauren A.; Pereira, Cliff; Baird, William M.

    2009-01-01

    The carcinogenic polycyclic aromatic hydrocarbon ns (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are widespread environmental pollutants, however their toxicological effects within a mixture is not established. We investigated the influence of diesel exhaust (DE) on B[a]P and DB[a,l]P-induced PAH-DNA adduct formation, metabolic activation, gene expression and 8-oxo-dG adduct levels in human breast epithelial cells (MCF-10A) in culture. Following 24 and 48 h, cells co-exposed to DE plus B[a]P exhibited a significant decrease in PAH-DNA adduct levels, compared with B[a]P alone, as determined by 33P-postlabeling combined with reversed-phase high performance liquid chromatography (HPLC). Cytochrome P450 (CYP) enzyme activity, as measured by the ethoxyresorufin O-deethylase (EROD) assay and CYP1B1 expression, significantly increased with co-exposure of DE plus DB[a,l]P, compared with DB[a,l]P alone. Aldo keto-reductase (AKR)1C1, AKR1C2,and AKR1C3 expression also significantly increased in cells exposed to DE plus PAH, compared with PAH exposure alone. Cell populations exhibiting 8-oxo-dG adducts significantly increased in response to exposure to B[a]P or DE plus B[a]P for 24 h, compared with vehicle control, as quantified by flow cytometry. These results suggest that complex mixtures may modify the carcinogenic potency of PAH by shifting the metabolic activation pathway from the production of PAH diol-epoxides to AKR pathway-derived metabolites. PMID:17612574

  14. Synchronous oscillation of the cytoplasmic Ca2+ concentration and membrane potential in cultured epithelial cells (Intestine 407).

    PubMed

    Yada, T; Oiki, S; Ueda, S; Okada, Y

    1986-06-16

    Cultured epithelial Intestine 407 cells exhibit regular oscillations of the membrane potential with repeated hyperpolarizations. These hyperpolarizations were inhibited not only by K+ channel blockers (tetraethylammonium and nonyltriethylammonium) but also by inhibitors of the Ca2+-activated K+ channel (quinine and quinidine). Using Ca2+-selective microelectrodes, cyclic increases in the cytosolic free Ca2+ concentration of more than 1 X 10(-6) M were found to coincide with the cyclic membrane hyperpolarizations. Thus, it appears that the potential oscillation is brought about by the oscillation of the intracellular free Ca2+ level which induces periodic activation of the Ca2+-dependent K+ channels. Neither the deprivation of extracellular Ca2+ nor the application of Ca2+ channel blockers (Co2+ and Ni2+) abolished the potential oscillation. Mitochondrial inhibitors (KCN, NaN3, antimycin A, FCCP and dinitrophenol) inhibited the potential oscillation, whereas glycolytic inhibitors (iodoacetic acid and NaF) had no effects. Caffeine and oxalate, which affect the microsomal Ca2+ transport, failed to exert any effect upon the potential oscillation. It is concluded that the cytosolic Ca2+ oscillation results from cyclic releases of Ca2+ from the intracellular storage site, which depends upon mitochondrial activities.

  15. Culture of prostate epithelial cells of the rhesus monkey on extracellular matrix substrate: influence of steroids and insulin-like growth factors.

    PubMed

    Udayakumar, T S; Jeyaraj, D A; Rajalakshmi, M; Sharma, R S

    1999-09-01

    Rhesus monkey prostate epithelial cells from the cranial lobe were isolated and cultured in flasks coated either with collagen IV or laminin. The effects of stromal cell medium, androgens and growth factors on cell number, thymidine incorporation and secretory activity were assessed. The results indicate that dihydrotestosterone (DHT) and androstenedione have stimulatory influences on cell proliferation and secretion in coated flasks. DHT was more effective in increasing cell number but the induction of secretory activity was similar with both steroids. The combination of IGF-I and -II resulted in inducing better cell proliferation and secretory activity than the individual IGFs but, of the two IGFs, IGF-I was more effective than IGF-II. DHT with IGFs was more potent in inducing proliferation, differentiation and secretion than androstenedione. Even in the absence of steroids or growth factors, colony formation and confluence occurred in coated flasks but cell differentiation and secretion only to a limited extent. In conclusion, we were able to establish an in vitro primary culture of prostate epithelial cells from rhesus monkey using extracellular matrix proteins, steroids and growth factors as additional supplements. This culture system may be useful to study prostate cell physiology and to identify drugs that can inhibit cell proliferation.

  16. Use of primary cultures of human bronchial epithelial cells isolated from cystic fibrosis patients for the pre-clinical testing of CFTR modulators.

    PubMed

    Neuberger, Timothy; Burton, Bill; Clark, Heather; Van Goor, Fredrick

    2011-01-01

    The use of human bronchial epithelial (HBE) cell cultures derived from the bronchi of CF patients offers the opportunity to study the effects of CFTR correctors and potentiators on CFTR function and epithelial cell biology in the native pathological environment. Cultured HBE cells derived from CF patients exhibit many of the morphological and functional characteristics believed to be associated with CF airway disease in vivo, including abnormal ion and fluid transport leading to dehydration of the airway surface and the loss of cilia beating. In addition, they can be generated in sufficient quantities to support routine lab testing of compound potency and efficacy and retain reproducible levels of CFTR function over time. Here we describe the development and validation of the CF HBE pharmacology model and its use to characterize, optimize, and select clinical candidates. It is expected that the pre-clinical testing of CFTR potentiators and correctors using epithelial cell cultures derived from CF patients will help to increase their likelihood of clinical efficacy.

  17. Effects of hydrogen peroxide on MAPK activation, IL-8 production and cell viability in primary cultures of human bronchial epithelial cells.

    PubMed

    Pelaia, Girolamo; Cuda, Giovanni; Vatrella, Alessandro; Gallelli, Luca; Fratto, Donatella; Gioffrè, Vincenza; D'Agostino, Bruno; Caputi, Mario; Maselli, Rosario; Rossi, Francesco; Costanzo, Francesco S; Marsico, Serafino A

    2004-09-01

    The airway epithelium is continuously exposed to inhaled oxidants, including airborne pollutants and cigarette smoke, which can exert harmful proinflammatory and cytotoxic effects. Therefore, the aim of our study was to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the signal transduction pathways activated by increasing concentrations (0.25, 0.5, and 1 mM) of hydrogen peroxide (H(2)O(2)), as well as their effects on IL-8 production and cell viability. The reported results show that H(2)O(2) elicited, in a concentration-dependent fashion, a remarkable increase in phosphorylation-dependent activation of mitogen-activated protein kinases (MAPKs), associated with a significant induction of IL-8 synthesis and a dramatically enhanced cell death. Pre-treatment of HBEC with MAPK inhibitors was able to significantly inhibit the effects of H(2)O(2) on IL-8 secretion, and to effectively prevent cell death. Therefore, these findings suggest that MAPKs play a key role as molecular transducers of the airway epithelial injury triggered by oxidative stress, as well as potential pharmacologic targets for indirect antioxidant intervention.

  18. Primary human epithelial cell culture system for studying interactions between female upper genital tract and sexually transmitted viruses, HSV-2 and HIV-1.

    PubMed

    Kaushic, Charu; Nazli, Aisha; Ferreira, Victor H; Kafka, Jessica K

    2011-10-01

    Evidence from clinical and epidemiological studies indicates that women are disproportionately susceptible to sexually transmitted viral infections. To understand the underlying biological basis for this increased susceptibility, more studies are needed to examine the acute events in the female reproductive tract following exposure to viruses during sexual transmission. The epithelial lining of the female reproductive tract is the primary barrier that sexually transmitted viruses, such as HIV-1 and HSV-2 need to infect or traverse, in order to initiate and establish productive infection. We have established an ex-vivo primary culture system to grow genital epithelial cells from upper reproductive tract tissues of women. Using these cultures, we have extensively examined the interactions between epithelial cells of the female genital tract and HSV-2 and HIV-1. In this review, we describe in detail the experimental protocol to grow these cultures, monitor their differentiation and inoculate with HSV-2 and HIV-1. Prospective use of these cultures to re-create the microenvironment in the reproductive tract is discussed.

  19. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.

    EPA Science Inventory

    Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling ...

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

    SciTech Connect

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  1. Inflammatory responses of a macrophage/epithelial cell co-culture model to mono and mixed infections with Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia.

    PubMed

    Bodet, Charles; Chandad, Fatiha; Grenier, Daniel

    2006-01-01

    Accumulated evidence points to Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as three major etiologic agents of chronic periodontitis. Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and the secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of a macrophage/epithelial cell co-culture model following mono or mixed infections with the above three periodontopathogens. An in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 monocytic cell line) was challenged with whole cells or lipopolysaccharides (LPS) of P. gingivalis, T. denticola, and T. forsythia, individually and in combination. Following stimulation, the production of interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), regulated on activation normal T cell expressed and secreted (RANTES), prostaglandin E2 (PGE2), and MMP-9 were quantified by enzyme-linked immunoassays. We observed that mono or mixed infections of the co-culture model induced the secretion of IL-1beta, IL-6, IL-8, PGE2, and MMP-9. P. gingivalis and T. forsythia induced an increase in RANTES secretion, whereas T. denticola alone or in combination resulted in a significant decrease in RANTES levels. All LPS challenges induced an increase in chemokine, MMP-9, and PGE2 production. No synergistic effect on the production of cytokines, chemokines, PGE2, and MMP-9 was observed for any of the bacterial or LPS mixtures tested. This study supports the view that P. gingivalis, T. denticola, and T. forsythia may induce high levels of pro-inflammatory mediators and MMP-9 in periodontal lesions, thus contributing to the progression of periodontitis.

  2. Effects of steroid hormones on differentiated glandular epithelial and stromal cells in a three dimensional cell culture model of the canine endometrium

    PubMed Central

    2013-01-01

    Background Oestrogens and progesterone have a significant impact on the endometrium during the canine oestrous cycle. Their receptors mediate plasma steroid hormone levels and are expressed in several endometrial cell types. Altered steroid receptor expression patterns are involved in serious uterine diseases; however the mechanisms of hormone action during pathogenesis in these tissues remain unclear. The development of 3D culture systems of canine endometrial cells provides an opportunity for the effects of steroid hormones to be quantitatively assessed in a more in vivo-like setting. The present study aimed to determine the effects of the steroid hormones 17β-estradiol (E) and progesterone (P) on the expression of the oestrogen and progesterone receptors (ER and PR), and on proliferative activity, in a 3D co-culture system of canine uterine origin, comprising differentiated endometrial glands, and stromal cells (SCs). Results Morphology, differentiation, and apical-basolateral polarity of cultured glandular epithelial cells (GECs) were comparable to those in native uterine tissue as assessed by immunohistochemistry using differentiation markers (β-catenin, laminin), lectin histochemistry, and transmission electron microscopy. Supplementation of our 3D-culture system with E (at 15, 30 and 100 pg/mL) resulted in constant levels of ER expression in GECs, but reduced expression levels in SCs. PR expression was reduced in both GECs and SCs following treatment with E. 3 ng/mL P resulted in increased ER expression in GECs, but a decrease in SCs. PR expression in GECs increased in all P-treated groups, whereas PRs in SCs decreased with the lowest and highest doses, but increased with the middle dose of treatment. Proliferative activity, assessed by Ki67 staining, remained below 1% in all assays and cell types. Conclusions The present study demonstrates the applicability of our 3D organotypic canine endometrium-derived culture system for cellular-level studies. 3D

  3. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

    PubMed

    Marcinkiewicz, Mariola M; Baker, Sandy T; Wu, Jichuan; Hubert, Terrence L; Wolfson, Marla R

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.

  4. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals

    PubMed Central

    Marcinkiewicz, Mariola M.; Baker, Sandy T.; Wu, Jichuan; Hubert, Terrence L.; Wolfson, Marla R.

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation—6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  5. Integrins and epithelial cell polarity.

    PubMed

    Lee, Jessica L; Streuli, Charles H

    2014-08-01

    Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell-matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical-basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity.

  6. Uptake and metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) by cultured renal tubular epithelial cells (RTEC)

    SciTech Connect

    Gordon, J.A.; Spector, A.A.

    1986-03-01

    To determine if 12-HETE, a lipoxygenase product that mediates inflammation and tissue injury, can interact with RTEC, confluent Madin Darby Canine Kidney (MDCK) cells were incubated for 2-16 hr with 1.0 ..mu..M (/sup 3/H)-12-HETE. Initial uptake of 12-HETE was rapid; at 16 hrs. 70% of the 12-HETE uptake was incorporated into phospholipids (PL). The distribution among the choline, ethanolamine, inositol, and serine PL was 36, 36, 20 and 8%, respectively. Incubation of MDCK cells with 0.5 to 5.0 ..mu..M (/sup 3/H)-12-HETE for 1 hr indicated linear uptake without evidence of saturation. Incubation with 1.0 ..mu..M 12-HETE and 0.25-10.0 ..mu..M arachidonic acid for 1 hr revealed no competition for uptake at the lower concentrations but a 40% reduction in 12-HETE uptake at 10.0 ..mu..M. Polarity of 12-HETE uptake was indicated by a preference of the basolateral surface over the apical surface by 1.4. After 2 hr, analysis of the medium by reverse phase HPLC revealed that 12-HETE was converted to three polar metabolites which eluted at 25.9, 29.4 and 31.3 min respectively; 12-HETE eluted at 37.5 min. The appearance of these polar metabolites was not prevented by ibuprofen (50 ..mu..M) nordihydroguaiaretic acid (30 ..mu..M), allopurinol (15 mM), or butylated hydroxytoluene (20 ..mu..M). These findings suggest that the lipoxygenase product 12-HETE may affect RTEC through incorporation into membrane PL and/or conversion to polar metabolites.

  7. Cultured Human Airway Epithelial Cells (Calu-3): A Model of Human Respiratory Function, Structure, and Inflammatory Responses

    PubMed Central

    Zhu, Yan; Chidekel, Aaron; Shaffer, Thomas H.

    2010-01-01

    This article reviews the application of the human airway Calu-3 cell line as a respiratory model for studying the effects of gas concentrations, exposure time, biophysical stress, and biological agents on human airway epithelial cells. Calu-3 cells are grown to confluence at an air-liquid interface on permeable supports. To model human respiratory conditions and treatment modalities, monolayers are placed in an environmental chamber, and exposed to specific levels of oxygen or other therapeutic modalities such as positive pressure and medications to assess the effect of interventions on inflammatory mediators, immunologic proteins, and antibacterial outcomes. Monolayer integrity and permeability and cell histology and viability also measure cellular response to therapeutic interventions. Calu-3 cells exposed to graded oxygen concentrations demonstrate cell dysfunction and inflammation in a dose-dependent manner. Modeling positive airway pressure reveals that pressure may exert a greater injurious effect and cytokine response than oxygen. In experiments with pharmacological agents, Lucinactant is protective of Calu-3 cells compared with Beractant and control, and perfluorocarbons also protect against hyperoxia-induced airway epithelial cell injury. The Calu-3 cell preparation is a sensitive and efficient preclinical model to study human respiratory processes and diseases related to oxygen- and ventilator-induced lung injury. PMID:20948883

  8. Effect of selenium nanoparticles with different sizes in primary cultured intestinal epithelial cells of crucian carp, Carassius auratus gibelio.

    PubMed

    Wang, Yanbo; Yan, Xuxia; Fu, Linglin

    2013-01-01

    Nano-selenium (Se), with its high bioavailability and low toxicity, has attracted wide attention for its potential application in the prevention of oxidative damage in animal tissues. However, the effect of nano-Se of different sizes on the intestinal epithelial cells of the crucian carp (Carassius auratus gibelio) is poorly understood. Our study showed that different sizes and doses of nano-Se have varied effects on the cellular protein contents and the enzyme activities of secreted lactate dehydrogenase, intracellular sodium potassium adenosine triphosphatase, glutathione peroxidase, and superoxide dismutase. It was also indicated that nano-Se had a size-dependent effect on the primary intestinal epithelial cells of the crucian carp. Thus, these findings may bring us a step closer to understanding the size effect and the bioavailability of nano-Se on the intestinal tract of the crucian carp.

  9. C22-bronchial and T7-alveolar epithelial cell lines of the immortomouse are excellent murine cell culture model systems to study pulmonary peroxisome biology and metabolism.

    PubMed

    Karnati, Srikanth; Palaniswamy, Saranya; Alam, Mohammad Rashedul; Oruqaj, Gani; Stamme, Cordula; Baumgart-Vogt, Eveline

    2016-03-01

    In pulmonary research, temperature-sensitive immortalized cell lines derived from the lung of the "immortomouse" (H-2k(b)-tsA58 transgenic mouse), such as C22 club cells and T7 alveolar epithelial cells type II (AECII), are frequently used cell culture models to study CC10 metabolism and surfactant synthesis. Even though peroxisomes are highly abundant in club cells and AECII and might fulfill important metabolic functions therein, these organelles have never been investigated in C22 and T7 cells. Therefore, we have characterized the peroxisomal compartment and its associated gene transcription in these cell lines. Our results show that peroxisomes are highly abundant in C22 and T7 cells, harboring a common set of enzymes, however, exhibiting specific differences in protein composition and gene expression patterns, similar to the ones observed in club cells and AECII in situ in the lung. C22 cells contain a lower number of larger peroxisomes, whereas T7 cells possess more numerous tubular peroxisomes, reflected also by higher levels of PEX11 proteins. Moreover, C22 cells harbor relatively higher amounts of catalase and antioxidative enzymes in distinct subcellular compartments, whereas T7 cells exhibit higher levels of ABCD3 and plasmalogen synthesizing enzymes as well as nuclear receptors of the PPAR family. This study suggest that the C22 and T7 cell lines of the immortomouse lung are useful models to study the regulation and metabolic function of the peroxisomal compartment and its alterations by paracrine factors in club cells and AECII.

  10. Human monocyte-derived dendritic cells from leukoreduction system chambers after plateletpheresis are functional in an in vitro co-culture assay with intestinal epithelial cells.

    PubMed

    Tiscornia, Inés; Sánchez-Martins, Viviana; Hernández, Ana; Bollati-Fogolín, Mariela

    2012-10-31

    The dendritic cells (DC) found in the intestine are involved both in the maintenance of tolerance towards commensal microbiota, and in the generation of protective immune responses against pathogens, thus contributing to gut immune homeostasis. There is an increasing interest in the use of lactic acid bacteria (LAB) as probiotics; among their beneficial effects we highlight the modulation of the immune system which is one of their fundamental properties. As these effects are strain-dependent, it is important to have in vitro systems that include DC and intestinal epithelial cells (IEC), which are crucial for intestinal homeostasis, to identify candidates by means of bacterial screening. Obtaining enough human cells, necessary to simultaneously test several bacteria, is a major challenge for researchers. In this study we analyzed the usefulness of the cellular fraction retained in leukoreduction system chambers following plateletpheresis (PP) as a source of DC. We compared the capacity of peripheral blood mononuclear cells (PBMC) from buffy coats (BC) or PP to generate DC using a short differentiation protocol. The functionality of the DC obtained was analyzed in co-cultures together with intestinal epithelial HT-29 cells, stimulating with LPS alone or with two LAB commonly used in the food industry, Streptococcus thermophilus and Lactobacillus delbrueckii. DC surface markers CD86, HLA-DR and cytokine production were measured. The behavior of DC derived from PP was similar to the behavior observed for DC derived from BC. When we tested the response of DC to bacteria, we found significant differences in cytokine secretion, especially for IL-10, suggesting that the system has the ability to discriminate LAB with different immunomodulatory properties. We also found that DC derived from both sources displayed a similar ability to phagocyte bacteria. In conclusion, we hereby propose a modification of the two-day protocol for obtaining human DC previously described, using

  11. Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

    NASA Technical Reports Server (NTRS)

    Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

    2011-01-01

    Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would

  12. Matrix metalloproteinase-9 expression is enhanced in renal parietal epithelial cells of zucker diabetic Fatty rats and is induced by albumin in in vitro primary parietal cell culture.

    PubMed

    Zhang, Yuanyuan; George, Jasmine; Li, Yun; Olufade, Rebecca; Zhao, Xueying

    2015-01-01

    As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy

  13. Phenotypic plasticity in normal breast derived epithelial cells

    PubMed Central

    2014-01-01

    Background Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. Results All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. Conclusions The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools. PMID:24915897

  14. TGF-β1 stimulates movement of renal proximal tubular epithelial cells in a three-dimensional cell culture via an autocrine TGF-β2 production.

    PubMed

    Luo, Deyi; Guan, Qiunong; Wang, Kunjie; Nguan, Christopher Y C; Du, Caigan

    2017-01-01

    TGF-βs are multifunctional cytokines, but their roles in human renal homeostasis are not fully understood. This study investigated the role of TGF-β1 in the movement of human renal proximal tubular epithelial cells (PTECs) in a three-dimensional (3D) model. HKC-8 cells, a human PTEC line, were grown in a 3D collagen culture system. Cell movement was observed under a microscope. The gene expression was examined using PCR Arrays or qRT-PCR, and protein levels by Western blot. Here, we showed that the tight junction structure formed between adjacent cells of a HKC-8 cell colony in 3D cultures, and TGF-β1 stimulated their movement, evidenced by the appearance of fingerlike pseudopodia in the leader cells at the edge of the colonies. The cell movement of these human PTECs was correlated with up-regulation of both MMP2 and MMP9 and down-regulation or inactivation of PLAUR and PTK2B. Analysis of TGF-β signaling targets confirmed autocrine production of TGF-β2 and its cleaving enzyme furin as well as SNAI1 by TGF-β1stimulation. Knockdown of TGF-β2 expression disrupted TGF-β1-stimulated PTEC invasiveness, which was correlated with the down-regulation of MMP2 and MMP9. In conclusion, the activation of TGF-β receptor autocrine signaling by up-regulated TGF-β2 may play a pivotal role in TGF-β1-induced human PTEC movement, which could be mediated at least by both MMP2 and MMP9.

  15. Comparison of several radiation effects in human MCF10A mammary epithelial cells cultured as 2D monolayers or 3D acinar stuctures in matrigel.

    PubMed

    Lin, Yu-Fen; Nagasawa, Hatsumi; Peng, Yuanlin; Chuang, Eric Y; Bedford, Joel S

    2009-06-01

    It has been argued that the cell-cell and cell-matrix interaction networks in normal tissues are disrupted by radiation and that this largely controls many of the most important cellular radiation responses. This has led to the broader assertion that individual cells in normal tissue or a 3D normal-tissue-like culture will respond to radiation very differently than the same cells in a 2D monolayer culture. While many studies have shown that, in some cases, cell-cell contact in spheroids of transformed or tumor cell lines can alter radiation responses relative to those for the same cells in monolayer cultures, a question remains regarding the possible effect of the above-mentioned disruption of signaling networks that operate more specifically for cells in normal tissues or in a 3D tissue-like context. To test the generality of this notion, we used human MCF-10A cells, an immortalized mammary epithelial cell line that produces acinar structures in culture with many properties of human mammary ducts. We compared the dose responses for these cells in the 2D monolayer and in 3D ductal or acinar structures. The responses examined were reproductive cell death, induction of chromosomal aberrations, and the levels of gamma-H2AX foci in cells after single acute gamma-ray doses and immediately after 20 h of irradiation at a dose rate of 0.0017 Gy/min. We found no significant differences in the dose responses of these cells in 2D or 3D growth conditions. While this does not mean that such differences cannot occur in other situations, it does mean that they do not generally or necessarily occur.

  16. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  17. Comparison of autosomal mutations in mouse kidney epithelial cells exposed to iron ions in situ or in culture.

    PubMed

    Turker, Mitchell S; Connolly, Lanelle; Dan, Cristian; Lasarev, Michael; Gauny, Stacey; Kwoh, Ely; Kronenberg, Amy

    2009-11-01

    Exposure to accelerated iron ions represents a significant health risk in the deep space environment because it induces mutations that can cause cancer. A mutation assay was used to determine the full spectrum of autosomal mutations induced by exposure to 2 Gy of 1 GeV/nucleon iron ions in intact kidney epithelium, and the results were compared with mutations induced in cells of a kidney epithelial cell line exposed in vitro. A molecular analysis for loss of heterozygosity (LOH) for polymorphic loci on chromosome 8, which harbors Aprt, demonstrated iron-ion induction of mitotic recombination, interstitial deletion, and discontinuous LOH events. Iron-ion-induced deletions were detected more readily with the in vitro assay, whereas discontinuous LOH was detected more readily in the intact kidney. The specific induction of discontinuous LOH in vivo suggests that this mutation pattern may serve as an indicator of genomic instability. Interestingly, the frequency of small intragenic events increased as a function of time after exposure, suggesting non-targeted effects. In total, the results demonstrate that 1 GeV/nucleon iron ions can elicit a variety of autosomal mutations and that the cellular microenvironment and the sampling time after exposure can influence the distribution of these mutations in epithelial cell populations.

  18. Use of a feline respiratory epithelial cell culture system grown at the air-liquid interface to characterize the innate immune response following feline herpesvirus 1 infection.

    PubMed

    Nelli, Rahul K; Maes, Roger; Kiupel, Matti; Hussey, Gisela Soboll

    2016-03-02

    Infection with feline herpesvirus-1 (FHV-1) accounts for 50% of viral upper respiratory diseases in domestic cats and is a significant cause of ocular diseases. Despite the clinical significance and high prevalence of FHV-1 infection, currently available vaccines cannot completely protect cats from infection and lifelong latency. FHV-1 infects via the mucous membranes and replicates in respiratory epithelial cells, but very little is known about the early innate immunity at this site. To address questions about immunity to FHV-1, feline respiratory epithelial cells cultured at air-liquid interface (ALI-FRECs) were established by collecting respiratory tracts from 6 healthy cats after euthanasia. Cells were isolated, cultured and characterized histologically and immunologically before infection with FHV-1. The expression of Toll-like receptors (TLRs), cytokine and chemokine responses were measured by real time PCR. ALI-FRECs morphologically resembled the natural airways of cats with multilayered columnar epithelial cells and cilia. Immunological properties of the natural airways were maintained in ALI-FRECs, as evidenced by the expression of TLRs, cytokines, chemokines, interferons, beta-defensins, and other regulatory genes. Furthermore, ALI-FRECs were able to support infection and replication of FHV-1, as well as modulate transcriptional regulation of various immune genes in response to infection. IL-1β and TNFα were increased in ALI-FRECs by 24hpi, whereas expression levels of IFN-α and TLR9 were not increased until 36hpi. In contrast, TLR3, GM-CSF and TGF-1β expression was down-regulated at 36hpi. The data presented show the development of a system ideal for investigating the molecular pathogenesis and immunity of FHV-1 or other respiratory pathogens.

  19. Modeling and Simulation of Mucus Flow in Human Bronchial Epithelial Cell Cultures - Part I: Idealized Axisymmetric Swirling Flow.

    PubMed

    Vasquez, Paula A; Jin, Yuan; Palmer, Erik; Hill, David; Forest, M Gregory

    2016-08-01

    A multi-mode nonlinear constitutive model for mucus is constructed directly from micro- and macro-rheology experimental data on cell culture mucus, and a numerical algorithm is developed for the culture geometry and idealized cilia driving conditions. This study investigates the roles that mucus rheology, wall effects, and HBE culture geometry play in the development of flow profiles and the shape of the air-mucus interface. Simulations show that viscoelasticity captures normal stress generation in shear leading to a peak in the air-mucus interface at the middle of the culture and a depression at the walls. Linear and nonlinear viscoelastic regimes can be observed in cultures by varying the hurricane radius and mean rotational velocity. The advection-diffusion of a drug concentration dropped at the surface of the mucus flow is simulated as a function of Peclet number.

  20. Modeling and Simulation of Mucus Flow in Human Bronchial Epithelial Cell Cultures – Part I: Idealized Axisymmetric Swirling Flow

    PubMed Central

    Vasquez, Paula A.; Jin, Yuan; Palmer, Erik; Hill, David; Forest, M. Gregory

    2016-01-01

    A multi-mode nonlinear constitutive model for mucus is constructed directly from micro- and macro-rheology experimental data on cell culture mucus, and a numerical algorithm is developed for the culture geometry and idealized cilia driving conditions. This study investigates the roles that mucus rheology, wall effects, and HBE culture geometry play in the development of flow profiles and the shape of the air-mucus interface. Simulations show that viscoelasticity captures normal stress generation in shear leading to a peak in the air-mucus interface at the middle of the culture and a depression at the walls. Linear and nonlinear viscoelastic regimes can be observed in cultures by varying the hurricane radius and mean rotational velocity. The advection-diffusion of a drug concentration dropped at the surface of the mucus flow is simulated as a function of Peclet number. PMID:27494700

  1. Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic.

    PubMed

    Gonzales, L W; Angampalli, S; Guttentag, S H; Beers, M F; Feinstein, S I; Matlapudi, A; Ballard, P L

    2001-01-01

    We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.

  2. Technical note: Isolation and characterization of porcine mammary epithelial cells.

    PubMed

    Dahanayaka, S; Rezaei, R; Porter, W W; Johnson, G A; Burghardt, R C; Bazer, F W; Hou, Y Q; Wu, Z L; Wu, G

    2015-11-01

    Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), β-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 μg/mL prolactin to culture medium for 3 d induced the production of β-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow.

  3. Effects of glucose on sorbitol pathway activation, cellular redox, and metabolism of myo-inositol, phosphoinositide, and diacylglycerol in cultured human retinal pigment epithelial cells.

    PubMed Central

    Thomas, T P; Porcellati, F; Kato, K; Stevens, M J; Sherman, W R; Greene, D A

    1994-01-01

    Sorbitol (aldose reductase) pathway flux in diabetes perturbs intracellular metabolism by two putative mechanisms: reciprocal osmoregulatory depletion of other organic osmolytes e.g., myo-inositol, and alterations in NADPH/NADP+ and/or NADH/NAD+. The "osmolyte" and "redox" hypotheses predict secondary elevations in CDP-diglyceride, the rate-limiting precursor for phosphatidylinositol synthesis, but through different mechanisms: the "osmolyte" hypothesis via depletion of intracellular myo-inositol (the cosubstrate for phosphatidylinositol-synthase) and the "redox" hypothesis through enhanced de novo synthesis from triose phosphates. The osmolyte hypothesis predicts diminished phosphoinositide-derived arachidonyl-diacylglycerol, while the redox hypothesis predicts increased total diacylglycerol and phosphatidic acid. In high aldose reductase expressing retinal pigment epithelial cells, glucose-induced, aldose reductase inhibitor-sensitive CDP-diglyceride accumulation and inhibition of 32P-incorporation into phosphatidylinositol paralleled myo-inositol depletion (but not cytoplasmic redox, that was unaffected by glucose) and depletion of arachidonyl-diacylglycerol. 3 mM pyruvate added to the culture medium left cellular redox unaltered, but stimulated Na(+)-dependent myo-inositol uptake, accumulation, and incorporation into phosphatidylinositol. These results favor myo-inositol depletion rather than altered redox as the primary cause of glucose-induced aldose reductase-related defects in phospholipid metabolism in cultured retinal pigment epithelial cells. Images PMID:8201009

  4. M-BAND analysis of chromosome aberration induced by Fe-ions in human epithelial cells cultured in 3-dimensional matrices

    NASA Astrophysics Data System (ADS)

    Hada, Megumi; Cucinotta, Francis A.; Wu, Honglu

    Energetic heavy ions pose a great health risk to astronauts in extended ISS and future lunar and Mars missions. High-LET heavy ions are particularly effective in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, which can be utilized as a biomarker for radiation insults. Previously, we had studied lowand high-LET radiationinduced chromosome aberrations in human epithelial cells cultured in 2-dimension (2D) using the multicolor banding fluorescence in situ hybridization (mBAND) technique. However, it has been realized that the biological response to radiation insult in a 2D cellular environment in vitro can differ significantly from the response in 3-dimension (3D) or at the actual tissue level. In this study, we cultured human epithelial cells in 3D to provide a more suitable model for human tissue. Human mammary epithelial cells (CH184B5F5/M10) were grown in Matrigel to form 3D structures, and exposed to Fe-ions at NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory or 137 Cs-gamma radiation source at the University of Texas MD Anderson Cancer Center. After exposure, cells were allowed to repair for 16hr before dissociation and subcultured at low density in 2D. G2 and metaphase chromosomes in the first cell cycle were collected using a chemical-induced premature chromosome condensation (PCC) technique, and chromosome aberrations were analyzed using mBAND technique. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of interchromosomal aberrations (translocation to unpainted chromosomes) and intrachromosomal aberrations (inversions and deletions within a single painted chromosome). Our data indicate a significant difference of the chromosome aberration yield between 2D and 3D cell cultures for gamma exposures, but not for Fe ion exposures

  5. Interphase Chromosome Conformation and Chromatin-chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Hada, Megumi; Wu, Honglu

    2014-01-01

    On a multi-mega base pair scale of the DNA, the arrangement of chromatin is non-random. In M10 epithelial cells, both telomere regions tend to be located towards the exterior of the chromosome domain, whereas the rest p-arm of the chromatin region towards the interior. In contrast, most of the q-arm of the chromatin is found in the peripheral of the domain. In lymphocytes, the p-arm chromatin regions towards the interior in close proximity with each other, whereas two q-arm regions are nearness in space. It indicates that G0 lymphocytes may lack secondary 3D chromatin folding. There chromatin folding patterns are consistent with our previous finding of non-random distribution of intra-chromosomal exchanges. In simulated microgravity conditions, the chromosome conformation may be altered and new regions in close proximity, especially to region 2 are suggested.

  6. Leptin upregulates beta3-integrin expression and interleukin-1beta, upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures.

    PubMed

    Gonzalez, R R; Leavis, P

    2001-10-01

    Human endometrium and endometrial epithelial cells (EECs) either cultured alone or cocultured with human embryos express leptin and leptin receptor. This study compares the effect of leptin with that of interleukin-1beta (IL-1beta) on the expression of beta3-EEC integrin, a marker of endometrial receptivity. Both cytokines increased the expression of beta3-EEC at concentrations in the range of 0.06-3 nM; however, leptin exhibited a significantly greater effect than IL-1beta. We also determined the regulatory effects of IL-1beta on leptin secretion and on the expression of leptin and leptin receptor at the protein level in both EEC and endometrial stromal cell (ESC) cultures. In EEC cultures, IL-1beta upregulated secretion of leptin and expression of both leptin and leptin receptors. No effect of IL-1beta was found in the ESC cultures. However, leptin exhibited marginal upregulation of leptin receptor. The upregulation of beta3-integrin and leptin/leptin receptor expression by IL-1beta in EEC cultures indicates that both cytokines may be implicated in embryonic-maternal cross-talk during the early phase of human implantation. Our present data also raise the possibility that leptin is an endometrial molecular effector of IL-1beta action on beta3-integrin upregulation. Thus, a new role for leptin in human reproduction as an autocrine/paracrine regulator of endometrial receptivity is proposed.

  7. Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro.

    PubMed

    Yuan, Chao; He, Qiang; Li, Jun-ming; Azzam, Mahmoud Mostafa; Lu, Jian-jun; Zou, Xiao-ting

    2015-06-01

    The present study evaluates the effects of embryonic age and proteolytic enzymes on the isolation and primary culture of chicken enterocyte and to establish an effective technique for chicken intestinal epithelial cell (IEC) cultivation. Fourteen-day-old, 16-day-old and 18-day-old embryos (average weight: 52.23 ± 0.76 g, 50.86 ± 0.99 g, 48.98 ± 1.03 g) were the source for preparation of enterocyte culture, and trypsin-ethylene diamine tetraacetic acid, collagenase, thermolysin and combination of collagenase and thermolysin were used for digestion medium. Optimal culture protocols were determined by qualitative assays of proliferation. Cells isolated by using 14-day-old embryo and collagenase obtain the best attachment and growth in culture, and the production of continuously growing IEC cultures. Thus, we conclude that the use of collagenase as a dissociating enzyme and 14-day-old embryo as a source can be advantageously applied to the isolation of chicken IEC and this method may be useful for various applications and basic studies of the intestinal tract concerning such objects as physiology, immunology and toxicology.

  8. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

    Ion channels in epithelial cells serve to move ions, and in some cases fluid, between compartments of the body. This function of the transfer of material is fundamentally different from that of the transfer of information, which is the main job of most channels in excitable cells. Nevertheless the basic construction of the channels is similar in many respects in the two tissue types. This chapter reviews the nature of channels in epithelia and discusses how their functions have evolved to accomplish the basic tasks for which they are responsible. I will focus on three channel types: epithelial Na+ channels, inward-rectifier K+ channels, and CFTR Cl- channels.

  9. Corrosive effects of fluoride on titanium: investigation by X-ray photoelectron spectroscopy, atomic force microscopy, and human epithelial cell culturing.

    PubMed

    Stájer, Anette; Ungvári, Krisztina; Pelsoczi, István K; Polyánka, Hilda; Oszkó, Albert; Mihalik, Erzsébet; Rakonczay, Zoltán; Radnai, Márta; Kemény, Lajos; Fazekas, András; Turzó, Kinga

    2008-11-01

    High fluoride (F(-)) concentrations and acidic pH impair the corrosion resistance of titanium (Ti). Effects of F(-)-containing caries-preventive prophylactic rinses, and gels on Ti were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Human epithelial cell attachment and proliferation were investigated by dimethylthiazol-diphenyl tetrazolium bromide (MTT) and protein content assays. Aqueous 1% NaF solution (3800 ppm F(-), pH 4.5) or high (12,500 ppm) F(-) content gel (pH 4.8) strongly corroded the surface and modified its composition. XPS revealed formation of a strongly bound F(-)-containing complex (Na(2)TiF(6)). AFM indicated an increase in roughness (R(a)) of the surfaces: 10-fold for the NaF solution and smaller for the gel or a mouthwash (250 ppm F(-), pH 4.4). MTT revealed that cell attachment was significantly increased by the gel, but was not disturbed by either the mouthwash or the NaF. Cell proliferation determined by MTT decreased significantly only for the NaF-treated samples; protein content assay experiments showed no such effect. This study indicates that epithelial cell culturing results can depend on the method used, and the adverse effects of a high F(-) concentration and low pH should be considered when prophylactic gels are applied by patients with Ti implants or other dental devices.

  10. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice

    PubMed Central

    Godin, Lindsay M.; Sandri, Brian J.; Wagner, Darcy E.; Meyer, Carolyn M.; Price, Andrew P.; Akinnola, Ifeolu; Weiss, Daniel J.; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases. PMID:26954258

  11. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice.

    PubMed

    Godin, Lindsay M; Sandri, Brian J; Wagner, Darcy E; Meyer, Carolyn M; Price, Andrew P; Akinnola, Ifeolu; Weiss, Daniel J; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.

  12. Interaction with epithelial cells modifies airway macrophage response to ozone.

    PubMed

    Bauer, Rebecca N; Müller, Loretta; Brighton, Luisa E; Duncan, Kelly E; Jaspers, Ilona

    2015-03-01

    The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

  13. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents.

    PubMed

    Zaccone, Eric J; Goldsmith, W Travis; Shimko, Michael J; Wells, J R; Schwegler-Berry, Diane; Willard, Patsy A; Case, Shannon L; Thompson, Janet A; Fedan, Jeffrey S

    2015-12-15

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance,we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity.We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥ 60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport,without affecting Cl- transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100-360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro.

  14. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents

    PubMed Central

    Zaccone, Eric J.; Goldsmith, W. Travis; Shimko, Michael J.; Wells, J.R.; Schwegler-Berry, Diane; Willard, Patsy A.; Case, Shannon L.; Thompson, Janet A.; Fedan, Jeffrey S.

    2016-01-01

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport, without affecting Cl− transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100–360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. PMID:26454031

  15. Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. III. Growth conditions of human thymic epithelial cells and immunomodulatory activities in their culture supernatant.

    PubMed Central

    Schreiber, L; Eshel, I; Meilin, A; Sharabi, Y; Shoham, J

    1991-01-01

    We report here on a new approach to the cultivation of human thymic epithelial (HTE) cells, which apparently allows more faithful preservation of cell function. This approach, previously developed by us for mouse thymic epithelial (MTE) cells, is based on the use of culture plates coated with extracellular matrix (ECM), and on the use of serum-free, growth factor-supplemented medium. The nutritional requirements of HTE and MTE are somewhat different. Although both are critically dependent on ECM and insulin, they differ in their dependency on other growth factors: selenium and transferrin are much more important for HTE cells, whereas epidermal growth factor and hydrocortisone play a more essential role in MTE cultures. The epithelial nature of the cultured cells is indicated by positive staining with anti-keratin antibodies and by the presence of desmosomes and tonofilaments. The ultrastructural appearance of the cells further suggests high metabolic and secretory activities, not usually found in corresponding cell lines. The culture supernatant (CS) of HTE cells exhibited a strong enhancing effect on thymocyte response to Con A stimulation, as measured by cell proliferation and lymphokine production. The effect was observed on both human and mouse thymocytes, but was much stronger in the homologous combination. Thymic factors tested in parallel did not have such a differential effect. The dose-effect relationships were in the form of a bell-shaped curve, with fivefold enhancement of response at the peak and a measurable effect even with 1:1000 dilution, when human thymocytes were used. The responding thymocytes were those which do not bind peanut agglutinin and are resistant to hydrocortisone. The culture system described here may have advantages for the in vitro study of thymic stromal cell function. Images Figure 1 Figure 3 Figure 4 PMID:1783421

  16. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

    Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

    1998-01-01

    Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial

  17. Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures.

    PubMed

    Fischer, William A; Chason, Kelly D; Brighton, Missy; Jaspers, Ilona

    2014-03-26

    Influenza viruses are global pathogens that infect approximately 10-20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent

  18. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

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

    PubMed Central

    Chao, Shih-Chun; Nien, Chan-Wei; Iacob, Codrin; Hu, Dan-Ning; Huang, Sheng-Chieh

    2016-01-01

    Dry eye is a common disorder characterized by deficiency of tear. Hyperosmoticity of tear stimulates inflammation and damage of ocular surface tissues and plays an essential role in the pathogenesis of dry eye. Cultured human corneal epithelial (CE) cells were used for the study of effects of lutein and hyperosmoticity on the secretion of IL-6 by CE cells. Cell viability of CE cells was not affected by lutein at 1–10 μM as determined by MTT assay. Hyperosmoticity significantly elevated the secretion of IL-6 by CE cells as measured by ELISA analysis. The constitutive secretion of IL-6 was not affected by lutein. Lutein significantly and dose-dependently inhibited hyperosmoticity-induced secretion of IL-6. Phosphorylated- (p)- p38 MAPK, p-JNK levels in cell lysates and NF-κB levels in cell nuclear extracts were increased by being exposed to hyperosmotic medium. JNK, p38, and NF-κB inhibitors decreased hyperosmoticity-induced secretion of IL-6. Lutein significantly inhibited hyperosmoticity-induced elevation of NF-κB, p38, and p-JNK levels. We demonstrated that lutein inhibited hyperosmoticity-induced secretion of IL-6 in CE cells through the deactivation of p38, JNK, and NF-κB pathways. Lutein may be a promising agent to be explored for the treatment of dry eye. PMID:27047687

  20. M-BAND Analysis of Chromosome Aberration Induced by Fe-Ions in Human Epithelial Cells Cultured in 3-Dimensional Matrices

    NASA Technical Reports Server (NTRS)

    Hada, M.; Cucinotta, F. A.; Wu, H.

    2008-01-01

    Energetic heavy ions pose a great health risk to astronauts in extended ISS and future lunar and Mars missions. High-LET heavy ions are particularly effective in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, which can be utilized as a biomarker for radiation insults. Previously, we had studied low- and high-LET radiation-induced chromosome aberrations in human epithelial cells cultured in 2-dimension (2D) using the multicolor banding fluorescence in situ hybridization (mBAND) technique. However, it has been realized that the biological response to radiation insult in a 2D cellular environment in vitro can differ significantly from the response in 3-dimension (3D) or at the actual tissue level. In this study, we cultured human epithelial cells in 3D to provide a more suitable model for human tissue. Human mammary epithelia cells (CH184B5F5/M10) were grown in Matrigel to form 3D structures, and exposed to Fe-ions at NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory or 137Cs-gamma radiation source at the University of Texas MD Anderson Cancer Center. After exposure, cells were allowed to repair for 16hr before dissociation and subcultued at low density in 2D. G2 and metaphase chromosomes in the first cell cycle were collected using a chemical-induced premature chromosome condensation (PCC) technique, and chromosome aberrations were analyzed using mBAND technique. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of interchromosomal aberrations (translocation to unpainted chromosomes) and intrachromosomal aberrations (inversions and deletions within a single painted chromosome). Our data indicate a significant difference of the chromosome aberration yield between 2D and 3D cell cultures for gamma exposures, but not for Fe ion exposures

  1. Effects of white light-emitting diode (LED) light exposure with different correlated color temperatures (CCTs) on human lens epithelial cells in culture.

    PubMed

    Xie, Chen; Li, Xiuyi; Tong, Jianping; Gu, Yangshun; Shen, Ye

    2014-01-01

    Cataract is the major cause for legal blindness in the world. Oxidative stress on the lens epithelial cells (hLECs) is the most important factor in cataract formation. Cumulative light-exposure from widely used light-emitting diodes (LEDs) may pose a potential oxidative threat to the lens epithelium, due to the high-energy blue light component in the white-light emission from diodes. In the interest of perfecting biosafety standards for LED domestic lighting, this study analyzed the photobiological effect of white LED light with different correlated color temperatures (CCTs) on cultured hLECs. The hLECs were cultured and cumulatively exposed to multichromatic white LED light with CCTs of 2954, 5624, and 7378 K. Cell viability of hLECs was measured by Cell Counting Kit-8 (CCK-8) assay. DNA damage was determined by alkaline comet assay. Intracellular reactive oxygen species (ROS) generation, cell cycle, and apoptosis were quantified by flow cytometry. Compared with 2954 and 5624 K LED light, LED light having a CCT of 7378 K caused overproduction of intracellular ROS and severe DNA damage, which triggered G2 /M arrest and apoptosis. These results indicate that white LEDs with a high CCT could cause significant photobiological damage to hLECs.

  2. Inverse-power-law behavior of cellular motility reveals stromal–epithelial cell interactions in 3D co-culture by OCT fluctuation spectroscopy

    PubMed Central

    Oldenburg, Amy L.; Yu, Xiao; Gilliss, Thomas; Alabi, Oluwafemi; Taylor, Russell M.; Troester, Melissa A.

    2015-01-01

    The progression of breast cancer is known to be affected by stromal cells within the local microenvironment. Here we study the effect of stromal fibroblasts on the in-place motions (motility) of mammary epithelial cells within organoids in 3D co-culture, inferred from the speckle fluctuation spectrum using optical coherence tomography (OCT). In contrast to Brownian motion, mammary cell motions exhibit an inverse power-law fluctuation spectrum. We introduce two complementary metrics for quantifying fluctuation spectra: the power-law exponent and a novel definition of the motility amplitude, both of which are signal- and position-independent. We find that the power-law exponent and motility amplitude are positively (p<0.001) and negatively (p<0.01) correlated with the density of stromal cells in 3D co-culture, respectively. We also show how the hyperspectral data can be visualized using these metrics to observe heterogeneity within organoids. This constitutes a simple and powerful tool for detecting and imaging cellular functional changes with OCT. PMID:26973862

  3. Toll-like receptor-2-activating bifidobacteria strains differentially regulate inflammatory cytokines in the porcine intestinal epithelial cell culture system: finding new anti-inflammatory immunobiotics.

    PubMed

    Fujie, Hitomi; Villena, Julio; Tohno, Masanori; Morie, Kyoko; Shimazu, Tomoyuki; Aso, Hisashi; Suda, Yoshihito; Shimosato, Takeshi; Iwabuchi, Noriyuki; Xiao, Jin-Zhong; Yaeshima, Tomoko; Iwatsuki, Keiji; Saito, Tadao; Numasaki, Muneo; Kitazawa, Haruki

    2011-10-01

    A total of 23 strains of bifidobacteria taxonomically belonging to five species were tested for their potent immunomodulatory effect using a combination of two methods: the NF-κB-reporter assay using a toll-like receptor 2-expressing transfectant (HEK(pTLR2) system) and the mitogenic assay using porcine Peyer's patches immunocompetent cells. Among the four preselected strains from different immunomodulatory groups, Bifidobacterium breve MCC-117 was able to efficiently modulate the inflammatory response triggered by enterotoxigenic Escherichia coli (ETEC) in a porcine intestinal epithelial (PIE) cell line. Moreover, using PIE cells and swine Peyer's patches immunocompetent cell co-culture system, we demonstrated that the immunoregulatory effect of B. breve MCC-117 was related to the capacity of the strain to influence PIE and immune cell interactions, leading to the stimulation of regulatory T cells. The results suggested that bifidobacteria that express high activity in both the HEK(pTLR2) and the mitogenic assays may behave like potential anti-inflammatory strains. The combination of the HEK(pTLR2) system, the evaluation of mitogenic activity and PIE cells will be of value for the development of new immunologically functional foods and feeds that could prevent inflammatory intestinal disorders. Although our findings should be proven in appropriate experiments in vivo, the results of the present work provide a scientific rationale for the use of B. breve MCC-117 to prevent ETEC-induced intestinal inflammation.

  4. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    SciTech Connect

    Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah; Cho, Jin Won; Lee, Joon H.

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful information

  5. The importance of the microenvironment in breast cancer progression: recapitulation of mammary tumorigenesis using a unique human mammary epithelial cell model and a three-dimensional culture assay

    PubMed Central

    Weaver, V.M.; Fischer, A.H.; Peterson, O.W.; Bissell, M.J.

    2010-01-01

    The extracellular matrix (ECM) is a dominant regulator of tissue development and homeostasis. “Designer microenvironments” in culture and in vivo model systems have shown that the ECM regulates growth, differentiation, and apoptosis in murine and human mammary epithelial cells (MEC) through a hierarchy of transcriptional events involving the intricate interplay between soluble and physical signaling pathways. Furthermore, these studies have shown that these pathways direct and in turn are influenced by the tissue structure. Tissue structure is directed by the cooperative interactions of the cell–cell and cell–ECM pathways and can be modified by stromal factors. Not surprisingly then, loss of tissue structure and alterations in ECM components are associated with the appearance and dissemination of breast tumors, and malignancy is associated with perturbations in cell adhesion, changes in adhesion molecules, and a stromal reaction. Several lines of evidence now support the contention that the pathogenesis of breast cancer is determined (at least in part) by the dynamic interplay between the ductal epithelial cells, the microenvironment, and the tissue structure (acini). Thus, to understand the mechanisms involved in carcinogenesis, the role of the microenvironment (ECM as well as the stromal cells) with respect to tissue structure should be considered and studied. Towards this goal, we have established a unique human MEC model of tumorigenesis, which in concert with a three-dimensional assay, recapitulates many of the genetic and morphological changes observed in breast cancer in vivo. We are currently using this system to understand the role of the microenvironment and tissue structure in breast cancer progression. PMID:9164652

  6. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  7. Inhibition of biliverdin reductase increases ANG II-dependent superoxide levels in cultured renal tubular epithelial cells

    PubMed Central

    Young, Shelby C.; Storm, Megan V.; Speed, Joshua S.; Kelsen, Silvia; Tiller, Chelsea V.; Vera, Trinity; Drummond, Heather A.

    2009-01-01

    Induction of heme oxygenase-1 (HO-1) in the renal medulla increases carbon monoxide and bilirubin production and decreases ANG II-mediated superoxide production. The goal of this study was to determine the importance of increases in bilirubin to the antioxidant effects of HO-1 induction in cultured mouse thick ascending loop of Henle (TALH) and inner medullary collecting duct (IMCD3) cells. Bilirubin levels were decreased by using small interfering RNAs (siRNAs) targeted to biliverdin reductase (BVR), which is the cellular enzyme responsible for the conversion of biliverdin to bilirubin. Treatment of cultured TALH or IMCD-3 cells with BVR siRNA (50 or 100 nM) resulted in an 80% decrease in the level of BVR protein and decreased cellular bilirubin levels from 46 ± 5 to 23 ± 4 nM (n = 4). We then determined the effects of inhibition of BVR on ANG II-mediated superoxide production. Superoxide production induced by ANG II (10−9 M) significantly increased in both TALH and IMCD-3 cells. Treatment of TALH cells with BVR siRNA resulted in a significant increase in ouabain-sensitive rubidium uptake from 95 ± 6 to 122 ± 5% control (n = 4, P < 0.05). Lastly, inhibition of BVR with siRNA did not prevent the decrease in superoxide levels observed in cells pretreated with the HO-1 inducer, hemin. We conclude that decreased levels of cellular bilirubin increase ANG II-mediated superoxide production and sodium transport; however, increases in bilirubin are not necessary for HO-1 induction to attenuate ANG II-mediated superoxide production. PMID:19759334

  8. Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro.

    PubMed

    Van Leer, Coretta; Stutz, Monika; Haeberli, André; Geiser, Thomas

    2005-12-01

    Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1beta, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 +/- 5.2% (p < 0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1beta-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix.

  9. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  10. Perifusion culture system for bovine embryos: improvement of embryo development by use of bovine oviduct epithelial cells, an antioxidant and polyvinyl alcohol.

    PubMed

    Lim, J M; Reggio, B C; Godke, R A; Hansel, W

    1997-01-01

    Three experiments were conducted in an attempt to improve a continuous flow-perifusion system capable of maintaining embryo development for long periods of time. Bovine embryos (8-16 cells) obtained from static co-culture with cumulus cells in a serum-free medium were perifused in an ACUSYST-S cell culture incubator. Culture chambers of the incubator consisted of a 0.2-mL unit (Chamber 1) connected to a 1.5-mL unit (Chamber 2), with the outflow from Chamber 1 routed to the inlet to Chamber 2. A bovine embryo culture medium supplemented with 3 mg mL-1 bovine serum albumin (BSA) and 25 mM HEPES was used as a perifusion culture medium (PCM). Embryos were perifused in Chamber 2 for 24, 48 and 72 h and further co-cultured in a static system up to 216 h after insemination. In Experiment 1, conditioning PCM with frozen-thawed bovine oviduct epithelial cells (BOEC) placed in Chamber 1 enhanced (P < 0.05) blastocyst formation of embryos in Chamber 2, after 24, 48 and 72 h of perifusion culture. The proportion of blastocysts was not further increased by placing BOEC in Chamber 2 along with the embryos. In Experiment 2, embryos were perifused with PCM conditioned with BOEC in Chamber 1 for 48 h or 72 h. A higher proportion of perifused embryos developed to the blastocyst stage after addition of 25 U mL-1 or 50 U mL-1 of superoxide dismutase (SOD) to PCM than in its absence. However, blastocyst formation of embryos perifused for 72 h was not increased after addition of 50 U mL-1 SOD compared with its absence. In Experiment 3, the proportions of morulae and blastocysts were not decreased by replacement of 3 mg mL-1 BSA with 1 mg mL-1 polyvinyl alcohol (PVA) in a BOEC-conditioned medium containing 50 U mL-1 SOD after perifusion for 48 h. In conclusion, PCM conditioning with BOEC and addition of an antioxidant to the perifusion medium improved the developmental capacity of perifused embryos. PVA is an adequate replacement for BSA in the perifusion medium.

  11. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line.

  12. The Apical Localization of Na(+), K(+)-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit.

    PubMed

    Lobato-Álvarez, Jorge A; Roldán, María L; López-Murillo, Teresa Del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

    2016-01-01

    Na(+), K(+)-ATPase, or the Na(+) pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β1 subunit of Na(+), K(+)-ATPase plays an important role in this mechanism because homotypic β1-β1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na(+) pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na(+), K(+)-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na(+) pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na(+), K(+)-ATPase in RPE cells depends on the expression of the β2 subunit.

  13. The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit

    PubMed Central

    Lobato-Álvarez, Jorge A.; Roldán, María L.; López-Murillo, Teresa del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

    2016-01-01

    Na+, K+-ATPase, or the Na+ pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β1 subunit of Na+, K+-ATPase plays an important role in this mechanism because homotypic β1-β1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na+ pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na+, K+-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na+ pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na+, K+-ATPase in RPE cells depends on the expression of the β2 subunit. PMID:27774068

  14. Apparatus for measuring the finite load-deformation behavior of a sheet of epithelial cells cultured on a mesoscopic freestanding elastomer membrane

    SciTech Connect

    Selby, John C.; Shannon, Mark A.

    2007-09-15

    Details are given for the design, calibration, and operation of an apparatus for measuring the finite load-deformation behavior of a sheet of living epithelial cells cultured on a mesoscopic freestanding elastomer membrane, 10 {mu}m thick and 5 mm in diameter. Although similar in concept to bulge tests used to investigate the mechanical properties of micromachined thin films, cell-elastomer composite diaphragm inflation tests pose a unique set of experimental challenges. Composite diaphragm (CD) specimens are extremely compliant (E<50 kPa), experience large displacements when subject to small inflation pressures ({approx}100 Pa), and must be continuously immersed in a bath of liquid culture medium during the acquisition of load-deformation measurements. Given these considerations, we have constructed an inflation apparatus consisting of an air-piston-cylinder pump integrated with a modular specimen mounting fixture that constitutes a horizontally semi-infinite reservoir of liquid culture medium. In a deformation-controlled inflation test, pressurized air is used to inflate a CD specimen into the liquid reservoir with minimum disturbance of the liquid-air interface. Piston displacements and absolute pump chamber air pressures are utilized as feedback to cycle the displaced (or inflated) CD volume V in a 0.05 Hz triangular or sinusoidal wave form (V{sub MIN}=0 {mu}l, V{sub MAX}{<=}40 {mu}l) while simultaneously recording the inflation pressure acting at the fixed boundary of the specimen, p(r=a). Using a carefully prescribed six-cycle inflation test protocol, the apparatus is shown to be capable of measuring the [V,p(r=a)] inflation response of a cell-elastomer CD with random uncertainties estimated at {+-}0.45 {mu}l and {+-}2.5 Pa, respectively.

  15. Apparatus for measuring the finite load-deformation behavior of a sheet of epithelial cells cultured on a mesoscopic freestanding elastomer membrane.

    PubMed

    Selby, John C; Shannon, Mark A

    2007-09-01

    Details are given for the design, calibration, and operation of an apparatus for measuring the finite load-deformation behavior of a sheet of living epithelial cells cultured on a mesoscopic freestanding elastomer membrane, 10 microm thick and 5 mm in diameter. Although similar in concept to bulge tests used to investigate the mechanical properties of micromachined thin films, cell-elastomer composite diaphragm inflation tests pose a unique set of experimental challenges. Composite diaphragm (CD) specimens are extremely compliant (E<50 kPa), experience large displacements when subject to small inflation pressures (approximately 100 Pa), and must be continuously immersed in a bath of liquid culture medium during the acquisition of load-deformation measurements. Given these considerations, we have constructed an inflation apparatus consisting of an air-piston-cylinder pump integrated with a modular specimen mounting fixture that constitutes a horizontally semi-infinite reservoir of liquid culture medium. In a deformation-controlled inflation test, pressurized air is used to inflate a CD specimen into the liquid reservoir with minimum disturbance of the liquid-air interface. Piston displacements and absolute pump chamber air pressures are utilized as feedback to cycle the displaced (or inflated) CD volume V in a 0.05 Hz triangular or sinusoidal wave form (V(MIN)=0 microl, V(MAX)cell-elastomer CD with random uncertainties estimated at +/-0.45 microl and +/-2.5 Pa, respectively.

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

    DTIC Science & Technology

    2012-04-01

    diverse transformed HMEC lines with defined genetic alterations may aid the identification of potential therapeutic treatments , including...human model systems to test potential therapeutics, could facilitate individualized treatment and possibly prevention. The main variables thought to...epithelial cells. Middle, corresponding cell culture models used in this study. Red, treatment or genetic manipulation used. Cell models are described in

  17. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing.

  18. Blue LED light exposure develops intracellular reactive oxygen species, lipid peroxidation, and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells.

    PubMed

    Nakanishi-Ueda, T; Majima, H J; Watanabe, K; Ueda, T; Indo, H P; Suenaga, S; Hisamitsu, T; Ozawa, T; Yasuhara, H; Koide, R

    2013-10-01

    The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm(2). The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm(2) exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm(2), respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm(2)).

  19. Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture.

    PubMed

    Gangalum, Rajendra K; Bhat, Ankur M; Kohan, Sirus A; Bhat, Suraj P

    2016-06-17

    Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.

  20. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.

  1. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    DOEpatents

    Stampfer, Martha R; Garbe, James C

    2015-02-24

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  2. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    SciTech Connect

    Stampfer, Martha R.; Garbe, James C.

    2016-06-28

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  3. Antioxidant Pre-Treatment Reduces the Toxic Effects of Oxalate on Renal Epithelial Cells in a Cell Culture Model of Urolithiasis

    PubMed Central

    Kizivat, Tomislav; Smolić, Martina; Marić, Ivana; Tolušić Levak, Maja; Smolić, Robert; Bilić Čurčić, Ines; Kuna, Lucija; Mihaljević, Ivan; Včev, Aleksandar; Tucak-Zorić, Sandra

    2017-01-01

    Urolithiasis is characterized by the formation and retention of solid crystals within the urinary tract. Kidney stones are mostly composed of calcium oxalate, which predominantly generates free radicals that are toxic to renal tubular cells. The aim of the study is to explore possible effects of antioxidant pre-treatment on inhibition of oxidative stress. Three cell lines were used as in vitro model of urolithiasis: MDCK I, MDCK II and LLC-PK1. Oxidative stress was induced by exposure of cells to sodium oxalate in concentration of 8 mM. In order to prevent oxidative stress, cells were pre-treated with three different concentrations of l-arginine and vitamin E. Oxidative stress was evaluated by determining the expression of superoxide dismutase (SOD), osteopontin (OPN), and by the concentration of glutathione (GSH). In all three cell lines, pre-treatment of antioxidants increased cell survival. Positive correlation of SOD and OPN expression as well as GSH concentration was observed in all groups of cells. Our results indicate that an antioxidant pre-treatment with l-arginine and vitamin E is able to hamper oxalate-induced oxidative stress in kidney epithelial cells and as such could play a role in prevention of urolithiasis. PMID:28125004

  4. Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells

    PubMed Central

    2004-01-01

    Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role

  5. Direct particle-to-cell deposition of coarse ambient particulate matter increases the production of inflammatory mediators from cultured human airway epithelial cells

    EPA Science Inventory

    Exposure of cultured cells to particulate matter air pollution is usually accomplished by collecting particles on a solid matrix, extracting the particles from the matrix, suspending them in liquid, and applying the suspension to cells grown on plastic and submerged in medium. Th...

  6. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  7. Cooperative Interactions During Human Mammary Epithelial Cell Immortalization

    DTIC Science & Technology

    2005-07-01

    Immortal Transformation of Cultured Human Mammary Epithelial Cells. Cellular Oncology, 26:248-251, 2004. Rodier , F., Kim, S-H., Nijjar, T., Yaswen, P...Promoter, Mol. Cell Biol.: 25:3923-3933, 2005. Goldstein, J, Rodier , F, Garbe, J, Stampfer, M, Campisi, J, Caspase-independent cytochrome c release is a

  8. Development of Na/sup +/-dependent hexose transport in cultured renal epithelial cells (LLC-PK/sub 1/)

    SciTech Connect

    Weiss, E.R.; Amsler, K.; Dawson, W.D.; Cook, J.S.

    1984-01-01

    A number of factors were explored to analyze how they interact to yield the increasing transport capacity in differentiating cell populations. These factors include the number of functional transporters in the population, the distribution of these transporters among the individual cells, the Na/sup +/ chemical gradient, the transmembrane potential, the pathways and activities of these pathways for efflux of glucoside, and cell-cell coupling between accumulating and non-accumulating cells. 35 references, 9 figures, 2 tables. (ACR)

  9. Inhibition of corneal epithelial cell migration by cadmium and mercury

    SciTech Connect

    Ubels, J.L.; Osgood, T.B. Medical Coll. of Wisconsin, Milwaukee )

    1991-02-01

    In a previous comparative study of corneal healing in fish, the authors observed that corneal epithelial healing occurs very rapidly in vivo in the marine teleost Myoxocephalus octodecimspinosus (longhorn sculpin) with a 6-mm diameter wound on the mammalian cornea. This rapid healing which permits prompt restoration of the epithelial barrier is apparently an adaptation to the large ionic and osmotic gradients between the environment and the intraocular fluids of the fish. These observations suggested that epithelial healing in the sculpin cornea might be useful model in aquatic biomedical toxicology if an in vitro method for measurement of healing rates could be developed. In this report the authors demonstrate that sculpin eyes maintained in short-term organ culture have a rapid corneal epithelial healing response and that this model can be used to demonstrate the toxic effects of heavy metals on epithelial cell migration.

  10. Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

    SciTech Connect

    Hirai, H.; Lochter, A.; Galosy, S.; Koshida, S.; Niwa, S.; Bissell, M.J.

    1997-10-13

    Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

  11. Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells – Relevance to Capsular Opacification

    PubMed Central

    Hojnik, Nataša; Filipič, Gregor; Lazović, Saša; Vesel, Alenka; Primc, Gregor; Mozetič, Miran; Hawlina, Marko; Petrovski, Goran; Cvelbar, Uroš

    2016-01-01

    Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO) due to remaining lens epithelial cells (LECs) by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation) at a highly targeted cell level compared to treatment on top of the medium (indirect treatment). Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity) to the posterior lens capsule and PCO formation. PMID:27832099

  12. Effects of the vegetable polyphenols epigallocatechin-3-gallate, luteolin, apigenin, myricetin, quercetin, and cyanidin in primary cultures of human retinal pigment epithelial cells

    PubMed Central

    Chen, Rui; Grosche, Antje; Reichenbach, Andreas; Wiedemann, Peter; Bringmann, Andreas; Kohen, Leon

    2014-01-01

    Purpose Vegetable polyphenols (bioflavonoids) have been suggested to represent promising drugs for treating cancer and retinal diseases. We compared the effects of various bioflavonoids (epigallocatechin-3-gallate [EGCG], luteolin, apigenin, myricetin, quercetin, and cyanidin) on the physiological properties and viability of cultured human retinal pigment epithelial (RPE) cells. Methods Human RPE cells were obtained from several donors within 48 h of death. Secretion of vascular endothelial growth factor (VEGF) was determined with enzyme-linked immunosorbent assay. Messenger ribonucleic acid levels were determined with real-time reverse transcription polymerase chain reaction. Cellular proliferation was investigated with a bromodeoxyuridine immunoassay, and chemotaxis was examined with a Boyden chamber assay. The number of viable cells was determined by Trypan Blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation enzyme-linked immunosorbent assay. The phosphorylation level of signaling proteins was revealed by western blotting. Results With the exception of EGCG, all flavonoids tested decreased dose-dependently the RPE cell proliferation, migration, and secretion of VEGF. EGCG inhibited the secretion of VEGF evoked by CoCl2-induced hypoxia. The gene expression of VEGF was reduced by myricetin at low concentrations and elevated at higher concentrations. Luteolin, apigenin, myricetin, and quercetin induced significant decreases in the cell viability at higher concentration, by triggering cellular necrosis. Cyanidin reduced the rate of RPE cell necrosis. Myricetin caused caspase-3 independent RPE cell necrosis mediated by free radical generation and activation of calpain and phospholipase A2. The myricetin- and quercetin-induced RPE cell necrosis was partially inhibited by necrostatin-1, a blocker of programmed necrosis. Most flavonoids tested diminished the phosphorylation levels of extracellular signal-regulated kinases 1/2 and Akt

  13. Concise review: limbal epithelial stem cell therapy: controversies and challenges.

    PubMed

    O'Callaghan, Anna R; Daniels, Julie T

    2011-12-01

    Limbal epithelial stem cells (LESCs) are a population of stem cells responsible for maintenance and repair of the corneal surface. Injury and disease can result in a deficiency of these stem cells, the vision affecting condition called limbal stem cell deficiency (LSCD) in which the cornea becomes opaque, vascularized, and inflamed. Cultured LESC therapy was first described in 1997;29:19231932-19231932.and LESCs cultured from either patients or donors have been used to successfully treat LSCD. In this review, some of the challenges and controversies associated with cultured LESC therapy will be discussed including alternative stem cell sources.

  14. Epithelial cell apoptosis facilitates Entamoeba histolytica infection in the gut.

    PubMed

    Becker, Stephen M; Cho, Kyou-Nam; Guo, Xiaoti; Fendig, Kirsten; Oosman, Mohammed N; Whitehead, Robert; Cohn, Steven M; Houpt, Eric R

    2010-03-01

    Entamoeba histolytica is the protozoan parasite that causes amebic colitis. The parasite triggers apoptosis on contact with host cells; however, the biological significance of this event during intestinal infection is unclear. We examined the role of apoptosis in a mouse model of intestinal amebiasis. Histopathology revealed that abundant epithelial cell apoptosis occurred in the vicinity of amoeba in histological specimens. Epithelial cell apoptosis occurred rapidly on co-culture with amoeba in vitro as measured by annexin positivity, DNA degradation, and mitochondrial dysfunction. Administration of the pan caspase inhibitor ZVAD decreased the rate and severity of amebic infection in CBA mice by all measures (cecal culture positivity, parasite enzyme-linked immunosorbent assay, and histological scores). Similarly, caspase 3 knockout mice on the resistant C57BL/6 background exhibited even lower cecal parasite antigen burden and culture positive rates than wild type mice. The permissive effect of apoptosis on infection could be tracked to the epithelium, in that transgenic mice that overexpressed Bcl-2 in epithelial cells were more resistant to infection as measured by cecal parasite enzyme-linked immunosorbent assay and histological scores. We concluded that epithelial cell apoptosis in the intestine facilitates amebic infection in this mouse model. The parasite's strategy for inducing apoptosis may point to key virulence factors, and therapeutic maneuvers to diminish epithelial apoptosis may be useful in amebic colitis.

  15. Quercetin Blocks Airway Epithelial Cell Chemokine Expression

    PubMed Central

    Nanua, Suparna; Zick, Suzanna M.; Andrade, Juan E.; Sajjan, Umadevi S.; Burgess, John R.; Lukacs, Nicholas W.; Hershenson, Marc B.

    2006-01-01

    Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a dietary flavonoid, is an inhibitor of phosphatidylinositol (PI) 3-kinase and potent antioxidant. We hypothesized that quercetin blocks airway epithelial cell chemokine expression via PI 3-kinase–dependent mechanisms. Pretreatment with quercetin and the PI 3–kinase inhibitor LY294002 each reduced TNF-α–induced IL-8 and monocyte chemoattractant protein (MCP)-1 (also called CCL2) expression in cultured human airway epithelial cells. Quercetin also inhibited TNF-α–induced PI 3-kinase activity, Akt phosphorylation, intracellular H2O2 production, NF-κB transactivation, IL-8 promoter activity, and steady-state mRNA levels, consistent with the notion that quercetin inhibits chemokine expression by attenuating NF-κB transactivation via a PI 3-kinase/Akt-dependent pathway. Quercetin also reduced TNF-α–induced chemokine secretion in the presence of the transcriptional inhibitor actinomycin D, while inducing phosphorylation of eukaryotic translation initiation factor (eIF)-2α, suggesting that quercetin attenuates chemokine expression by post-transcriptional as well as transcriptional mechanisms. Finally, we tested the effects of quercetin in cockroach antigen–sensitized and –challenged mice. These mice show MCP-1–dependent airways hyperresponsiveness and inflammation. Quercetin significantly reduced lung MCP-1 and methacholine responsiveness. We conclude that quercetin blocks airway cell chemokine expression via transcriptional and post-transcriptional pathways. PMID:16794257

  16. Proinflammatory responses of a hTERT-transformed, immortalized line of cultured bovine mammary epithelial cells (BME)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Primary cultures BME were generated from healthy mammary glands as described (Vet Immunol Immunopath 101(3-4):191-202, 2004). Towards immortalization, BME from four cows were pooled and transfected with pCI neo-hEST2-HA , a human telomerase segment containing a neomycin/Geneticin resistance select...

  17. Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils.

    PubMed

    Hosoki, Koa; Kainuma, Keigo; Toda, Masaaki; Harada, Etsuko; Chelakkot-Govindalayathila, Ayshwarya-Lakshmi; Roeen, Ziaurahman; Nagao, Mizuho; D'Alessandro-Gabazza, Corina N; Fujisawa, Takao; Gabazza, Esteban C

    2014-07-04

    Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.

  18. Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture.

    PubMed

    Leighton, J

    1994-09-01

    The biology of animal cells in culture is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three-dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and of morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three-dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial tissues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin permeable transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.

  19. Airway epithelial cell wound repair mediated by alpha-dystroglycan.

    PubMed

    White, S R; Wojcik, K R; Gruenert, D; Sun, S; Dorscheid, D R

    2001-02-01

    Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.

  20. Metformin inhibits the proliferation of benign prostatic epithelial cells

    PubMed Central

    Ge, Rongbin; Li, Jijun; Johnson, Cameron W.; Rassoulian, Cyrus; Olumi, Aria F.

    2017-01-01

    Objective Benign prostatic hyperplasia (BPH) is the most common proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies have shown that diabetes significantly increases the risk of developing BPH, although whether anti-diabetic medications preventing the development of BPH remains to be defined. We have previously found that stromally expressed insulin-like growth factor 1 (IGF-1) promotes benign prostatic epithelial cell proliferation through paracrine mechanisms. Here, we seek to understand if metformin, a first line medication for the treatment of type 2 diabetes, inhibits the proliferation of benign prostatic epithelial cells through reducing the expression of IGF-1 receptor (IGF-1R) and regulating cell cycle. Methods BPE cell lines BPH-1 and P69, murine fibroblasts3T3 and primary human prostatic fibroblasts were cultured and tested in this study. Cell proliferation and the cell cycle were analyzed by MTS assay and flow cytometry, respectively. The expression of IGF-1R was determined by western-blot and immunocytochemistry. The level of IGF-1 secretion in culture medium was measured by ELISA. Results Metformin (0.5-10mM, 6-48h) significantly inhibited the proliferation of BPH-1 and P69 cells in a dose-dependent and time-dependent manner. Treatment with metformin for 24 hours lowered the G2/M cell population by 43.24% in P69 cells and 24.22% in BPH-1 cells. On the other hand, IGF-1 (100ng/mL, 24h) stimulated the cell proliferation (increased by 28.81% in P69 cells and 20.95% in BPH-1 cells) and significantly enhanced the expression of IGF-1R in benign prostatic epithelial cells. Metformin (5mM) abrogated the proliferation of benign prostatic epithelial cells induced by IGF-1. In 3T3 cells, the secretion of IGF-1 was significantly inhibited by metformin from 574.31pg/ml to 197.61pg/ml. The conditioned media of 3T3 cells and human prostatic fibroblasts promoted the proliferation of epithelial cells and the

  1. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

    Song, Chun-Hua; Liu, Zhi-Qiang; Huang, Shelly; Zheng, Peng-Yuan; Yang, Ping-Chang

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barrier function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.

  2. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    SciTech Connect

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  3. Development of human epithelial cell systems for radiation risk assessment

    NASA Astrophysics Data System (ADS)

    Yang, C. H.; Craise, L. M.

    1994-10-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  4. Development of human epithelial cell systems for radiation risk assessment

    NASA Technical Reports Server (NTRS)

    Yang, C. H.; Craise, L. M.

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  5. Effect of mineral particles containing iron on primary cultures of rabbit tracheal epithelial cells: possible implication of oxidative stress.

    PubMed Central

    Guilianelli, C; Baeza-Squiban, A; Boisvieux-Ulrich, E; Houcine, O; Zalma, R; Guennou, C; Pezerat, H; Marano, F

    1993-01-01

    Environmental mineral particles such as asbestos are responsible for numerous respiratory diseases. In addition to effects related to their geometry, particles are now assumed to act by triggering an oxidative stress process. Iron-containing particles, in particular, can produce oxygen-activated species by oxidizing their iron. To evaluate the involvement of iron-containing particles in respiratory diseases, three mineral particles (chrysotile, nemalite, and hematite) were tested in primary cultures of tracheal epithelium. Because of the ciliary beat, the three mineral particles were quickly concentrated at the periphery of the mucociliary epithelium, reconstituted in vitro where they induced cellular lesions. Endocytosis of the three types of particles was observed. Cytotoxicity studies have indicated that among the tested particles, the most cytostatic after 24 hr of treatment was the one that contained more Fe2+ available on the surface, nemalite. Moreover, the effect of nemalite was reduced by pretreatment with desferrioxamine. As mineral particles, especially asbestos, are suspected to induce squamous metaplasia, we chose to study two specific transformations of the epithelium: the expression of cytokeratin-13 and the formation of cross-linked envelopes. Under our culture conditions, nemalite and chrysotile increased the expression of the cytokeratin-13, a specific marker of squamous metaplasia, whereas nemalite was the only particle able to strongly induce the formation of cross-linked envelopes. Nemalite was the most cytostatic particle and the most efficient at inducing squamous metaplasia. Measures of oxidizing power by electron-spin resonance revealed that nemalite produced the most oxygen-activated species.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1. Figure 2. a Figure 2. b Figure 2. c Figure 2. d Figure 2. e Figure 2. f Figure 3. a Figure 3. b Figure 3. c Figure 3. d Figure 4. Figure 5. a Figure 5. b Figure 5. c Figure 5. d Figure 6. PMID:8119255

  6. DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

    PubMed

    Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

    2014-04-01

    DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

  7. Development of Methodology to Maintain Primary Cultures of Normal and Malignant Human Prostatic Epithelial Cells In Vivo

    DTIC Science & Technology

    2008-02-01

    grafting site. Several xenotransplantation models were developed based on severe combined immunodeficient (SCID) mice and their derivative, the non... xenotransplantation of human plasmacytoid dendritic cell precursors and human peripheral blood lymphocytes for the development of a severe acute graft-vs

  8. Development of Methodology to Maintain Primary Cultures of Normal and Malignant Human Prostatic Epithelial Cells in Vivo

    DTIC Science & Technology

    2006-02-01

    xenotransplantation models were developed based on severe combined immunodeficient (SCID) mice and their derivative, the non-obese diabetic (NOD)/SCID mouse model. The...all T and B cells and NK function. This novel immunodeficient mouse proved to be useful for studying xenotransplantation of human plasmacytoid dendritic

  9. Bone marrow-derived lung epithelial cells.

    PubMed

    Krause, Diane S

    2008-08-15

    Bone marrow-derived cells can take on the phenotype of epithelial cells and express epithelial-specific genes in multiple organs. Here, we focus on recent data on the appearance of marrow-derived epithelial cells in the adult lung. These findings have garnered significant skepticism because in most cases marrow-derived epithelial cells are very rare, the marrow cell of origin is not known, the techniques for detection have needed improvement, and there seem to be multiple mechanisms by which this occurs. Recent studies have focused on these concerns. Once these important concerns are addressed, further studies on the function(s) of these cells will need to be performed to determine whether this engraftment has any clinical significance-either beneficial or detrimental.

  10. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    PubMed

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

  11. Phenotypic modification of human airway epithelial cells in air-liquid interface culture induced by exposure to the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

    PubMed

    Carson, Johnny L; Brighton, Luisa E; Jaspers, Ilona

    2015-04-01

    The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen. We used an air-liquid interface epithelial cell culture system to model changes associated with NNK exposure relative to pathologies documented in human tobacco-related illnesses. Although in vitro systems exhibit certain limitations, they often offer accentuation of subtle pathologies. While the distribution of cell types in control cultures typically favors the ciliated cell phenotype, NNK-exposed cultures transitioned to non-ciliated cell phenotypes as well as reflecting features consistent with squamous metaplasia. We conclude that NNK impacts normal growth and differentiation of human airway epithelium in a short interval of time in vitro.

  12. The Role of E-Cadherin in Maintaining the Barrier Function of Corneal Epithelium after Treatment with Cultured Autologous Oral Mucosa Epithelial Cell Sheet Grafts for Limbal Stem Deficiency

    PubMed Central

    Hoft, Richard H.; Wood, Andrew; Oliva, Joan; Niihara, Hope; Makalinao, Andrew; Thropay, Jacquelyn; Pan, Derek; Tiger, Kumar; Garcia, Julio; Laporte, Amanda; French, Samuel W.; Niihara, Yutaka

    2016-01-01

    The role of E-cadherin in epithelial barrier function of cultured autologous oral mucosa epithelial cell sheet (CAOMECS) grafts was examined. CAOMECS were cultured on a temperature-responsive surface and grafted onto rabbit corneas with Limbal Stem Cell Deficiency (LSCD). E-cadherin levels were significantly higher in CAOMECS compared to normal and LSCD epithelium. Beta-catenin colocalized with E-cadherin in CAOMECS cell membranes while phosphorylated beta-catenin was significantly increased. ZO-1, occludin, and Cnx43 were also strongly expressed in CAOMECS. E-cadherin and beta-catenin localization at the cell membrane was reduced in LSCD corneas, while CAOMECS-grafted corneas showed a restoration of E-cadherin and beta-catenin expression. LSCD corneas did not show continuous staining for ZO-1 or for Cnx43, while CAOMECS-grafted corneas showed a positive expression of ZO-1 and Cnx43. Cascade Blue® hydrazide did not pass through CAOMECS. Because E-cadherin interactions are calcium-dependent, EGTA was used to chelate calcium and disrupt cell adhesion. EGTA-treated CAOMECS completely detached from cell culture surface, and E-cadherin levels were significantly decreased. In conclusion, E cadherin high expression contributed to CAOMECS tight and gap junction protein recruitment at the cell membrane, thus promoting cellular adhesion and a functional barrier to protect the ocular surface. PMID:27777792

  13. Proliferation of rhesus ovarian surface epithelial cells in culture: Lack of mitogenic response to steroid or gonadotropic hormones

    SciTech Connect

    Wright, Jay W.; Toth-Fejel, Suellen; Stouffer, Richard L.; Rodland, Karin D.

    2002-06-30

    Ovarian cancer is the most lethal gynecological cancer and approximately 90% of ovarian cancers derive from the ovarian surface epithelium (OSE), yet the biology of the OSE is poorly understood. Factors associated with increased risk of non-hereditary ovarian cancer include the formation of inclusion cysts, effects of reproductive hormones cytokeratin, vimentin, N-cadherin, E-cadherin, estrogen receptor-a, and progesterone receptor. We show that these cells activate MAP Kinase and proliferate in response to extracellular calcium, as do human and rat OSE. In contrast, the gonadotropic hormones FSH (4-400 IU/L), LH (8.5-850 IU/l), and hCG (10-1000 IU/l) fail to stimulate proliferation. We find that concentrations of progesterone and estrogen normally present in follicles just prior to ovulation ( ~1000 ng/ml) significantly decrease the number of mitotically active RhOSE cells as determined by PCNA labelling, total cell count, and 3H-thymidine uptake, while lower steroid concentrations have no effect.

  14. Akt-mediated transforming growth factor-β1-induced epithelial-mesenchymal transition in cultured human esophageal squamous cancer cells.

    PubMed

    Xuan, X; Zeng, Q; Li, Y; Gao, Y; Wang, F; Zhang, H; Wang, Z; He, H; Li, S

    2014-06-01

    Epithelial-mesenchymal transition (EMT) has a crucial role during embryonic development and has also come under intense scrutiny as a mechanism through which esophageal squamous cell cancer (ESCC) progresses to become metastatic. Transforming growth factor beta (TGF-β)-mediated EMT has been observed in a variety of cell types and has been identified as the main inducer of EMT in many types of cancer. Akt activity is involved in TGF-β-mediated EMT; however, its precise relationship and role in EMT in ESCC has not been well explained to date. Our data demonstrated that in human ESCC tissues Akt and its activated form, phosphorylated-Akt (p-Akt), were overexpressed; in addition, Akt and p-Akt were negatively correlated with epithelial cadherin (E-cadherin). In EC-9706 cells, exogenous TGF-β1 could induce EMT and at the same time could increase the EC-9706 cell invasive and metastatic ability. Moreover, Akt knockdown by small-interfering RNA could attenuate the EMT induced by TGF-β1 by increasing the epithelial marker E-cadherin and decreasing the mesenchymal marker Vimentin. Silencing Akt expression could decrease the migration ability of EC-9706 cells efficiently. In short, Akt is likely to have a more important role in the EMT induced by TGF-β1 in EC-9706 and may contribute to the invasive and metastatic ability of EC-9706. Akt may be an effective therapeutic in advanced and metastatic ESCC.

  15. Cell line-dependent cytotoxicity of poly(isobutylcyanoacrylate) nanoparticles coated with chitosan and thiolated chitosan: Insights from cultured human epithelial HeLa, Caco2/TC7 and HT-29/MTX cells.

    PubMed

    Pradines, Bénédicte; Lievin-Le Moal, Vanessa; Vauthier, Christine; Ponchel, Gilles; Loiseau, Philippe M; Bouchemal, Kawthar

    2015-08-01

    Nanoparticles composed of poly(isobutylcyanoacrylate) core coated with a mixture of chitosan and thiolated chitosan have already shown promising results in terms of mucoadhesion and permeation enhancement properties of pharmaceutical active drugs delivered via mucosal routes. In the present work, the cytotoxicity of these nanoparticles was first investigated using direct contact assay on undifferentiated human cervix epithelial HeLa cells. The results showed strong toxicity in HeLa cells for the two investigated concentrations 25 and 50 μg/mL. The cytotoxic effect was mainly attributed to the poly(isobutylcyanoacrylate) core since no significant differences in nanoparticle cytotoxicity were reported when nanoparticle shell composition was modified by adding chitosan or thiolated chitosan. In contrast, lower nanoparticle toxicity was reported using human fully-differentiated enterocyte-like Caco-2/TC7, and fully-differentiated mucus-secreting HT-29/MTX cells forming monolayer in culture mimicking an intestinal epithelial barrier. This study demonstrated that the toxicity of poly(isobutylcyanoacrylate) nanoparticles is highly cell line-dependent.

  16. Fibrin glue inhibits migration of ocular surface epithelial cells.

    PubMed

    Yeung, A M; Faraj, L A; McIntosh, O D; Dhillon, V K; Dua, H S

    2016-10-01

    PurposeFibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro.MethodsCorneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed.ResultsExplants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14-16 for explants with fibrin glue.ConclusionsFibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy.

  17. Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate.

    PubMed

    Pennington, Britney O; Clegg, Dennis O; Melkoumian, Zara K; Hikita, Sherry T

    2015-02-01

    Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies.

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

    PubMed

    Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

    2015-01-01

    Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

  19. Small molecule and RNAi induced phenotype transition of expanded and primary colonic epithelial cells

    PubMed Central

    Sharbati, Jutta; Hanisch, Carlos; Pieper, Robert; Einspanier, Ralf; Sharbati, Soroush

    2015-01-01

    Recent progress in mammalian intestinal epithelial cell culture led to novel concepts of tissue modeling. Especially the development of phenotypically stable cell lines from individual animals enables an investigation of distinct intestinal loci and disease states. We here report primary and prolonged culture of normal porcine epithelial cells from colon for cell line development. In addition, a novel primary three-dimensional intestinal culture system is presented, which generated organoids composed of a highly polarized epithelial layer lining a core of subepithelial tissue. Cellular characterization of monolayer cell lines revealed epithelial identity and pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and chemical approaches to induce epithelial differentiation in generated cell lines by targeting promoters of epithelial to mesenchymal transition (EMT). By in silico prediction and ectopic expression, miR-147b was proven to be a potent trigger of intestinal epithelial cell differentiation. Our results outline an approach to generate phenotypically stable cell lines expanded from primary colonic epithelial cultures and demonstrate the relevance of miR-147b and chemical inhibitors for promoting epithelial differentiation features. PMID:26223582

  20. Mechanobiology in Lung Epithelial Cells: Measurements, Perturbations, and Responses

    PubMed Central

    Waters, Christopher M.; Roan, Esra; Navajas, Daniel

    2015-01-01

    Epithelial cells of the lung are located at the interface between the environment and the organism and serve many important functions including barrier protection, fluid balance, clearance of particulate, initiation of immune responses, mucus and surfactant production, and repair following injury. Because of the complex structure of the lung and its cyclic deformation during the respiratory cycle, epithelial cells are exposed to continuously varying levels of mechanical stresses. While normal lung function is maintained under these conditions, changes in mechanical stresses can have profound effects on the function of epithelial cells and therefore the function of the organ. In this review, we will describe the types of stresses and strains in the lungs, how these are transmitted, and how these may vary in human disease or animal models. Many approaches have been developed to better understand how cells sense and respond to mechanical stresses, and we will discuss these approaches and how they have been used to study lung epithelial cells in culture. Understanding how cells sense and respond to changes in mechanical stresses will contribute to our understanding of the role of lung epithelial cells during normal function and development and how their function may change in diseases such as acute lung injury, asthma, emphysema, and fibrosis. PMID:23728969

  1. [Methotrexate as inducer of proinflammatory cytokines by epithelial cells].

    PubMed

    Morón-Medina, Alejandra; Viera, Ninoska; de Morales, Thaís Rojas; Alcocer, Sirley; Bohorquez, Dinorath

    2014-03-01

    Methotrexate (MTX), a drug commonly used in childhood cancer, has also been indicated as a cytotoxic agent of the oral mucosa, which can trigger the inflammatory process and increase the vascularity of epithelial tissues during the early stages of oral mucositis. The aim of this study was to determine the production of proinflammatory cytokines IL-1beta, IL-6 y TNF-alpha in epithelial cell cultures treated with MTX. Epithelial cells of human larynx, obtained from the cell line Hep-2, were cultured with different doses of MTX during different incubation times. The drug cytotoxicity was analyzed by means of the colorimetric test, which is based on the metabolic reduction of the bromide of 3-(4, 5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol (MTT); and the proinflammatory cytokines production by the test enzyme-linked immunosorbent assay (ELISA). Cultures of HEp-2 cells showed increased production of proinflammatory cytokines at 72 hours with 0.32 microM of MTX. These results suggest that depending on the dose and exposure time, MTX alters the physiology of human epithelial cells, which may play an important role during the phases of initiation and development of oral mucositis.

  2. Claudin-4 forms a paracellular barrier, revealing the interdependence of claudin expression in the loose epithelial cell culture model opossum kidney cells.

    PubMed

    Borovac, Jelena; Barker, Reid S; Rievaj, Juraj; Rasmussen, Andrew; Pan, Wanling; Wevrick, Rachel; Alexander, R Todd

    2012-12-15

    The effect of claudins on paracellular fluxes has been predominantly studied in either Madin-Darby canine kidney (MDCK) or LLCPK cells. Neither model system has a very low transepithelial resistance (TER) as observed in leaky epithelia. Moreover, results from one model system are not always consistent with another. Opossum kidney (OK) cells form tight junctions yet have a very low TER. We therefore set out to characterize the paracellular transport properties of this cell culture model. Ussing chamber dilution potential measurements revealed that OK cells exhibit a very low TER (11.7 ± 1.4 Ω·cm(2)), slight cation selectivity (P(Na)/P(Cl) = 1.10 ± 0.01), and the Eisenman permeability sequence IV; the permeability of monovalent cations ranking K(+) > Cs(+) > Rb(+) > Na(+) > Li(+). Quantitative real-time PCR studies found that OK cells endogenously express claudin-4 > -1 > -6 > -20 > -9 > -12 > -11 > -15. Overexpression of claudin-4 significantly increased TER, decreased Na(+) and Cl(-) permeability, and increased levels of claudin-1, -6, and -9 mRNA. Knockdown of claudin-4 in the overexpressing cells significantly decreased TER without altering claudin expression; thus claudin-4 forms a barrier in OK cells. Knockdown of endogenous claudin-4 decreased claudin-1, -9, and -12 expression without altering TER. Claudin-2 overexpression decreased TER, significantly increased Na(+) and Cl(-) permeability, and decreased claudin-12 and -6 expression. Together these results demonstrate that claudin expression is tightly coupled in OK cells.

  3. Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro

    PubMed Central

    Zheng, Dahai; Soh, Boon-Seng; Yin, Lu; Hu, Guangan; Chen, Qingfeng; Choi, Hyungwon; Han, Jongyoon; Chow, Vincent T. K.; Chen, Jianzhu

    2017-01-01

    Club cells are known to function as regional progenitor cells to repair the bronchiolar epithelium in response to lung damage. By lineage tracing in mice, we have shown recently that club cells also give rise to alveolar type 2 cells (AT2s) and alveolar type 1 cells (AT1s) during the repair of the damaged alveolar epithelium. Here, we show that when highly purified, anatomically and phenotypically confirmed club cells are seeded in 3-dimensional culture either in bulk or individually, they proliferate and differentiate into both AT2- and AT1-like cells and form alveolar-like structures. This differentiation was further confirmed by transcriptomic analysis of freshly isolated club cells and their cultured progeny. Freshly isolated club cells express Sca-1 and integrin α6, markers commonly used to characterize lung stem/progenitor cells. Together, current study for the first time isolated highly purified club cells for in vitro study and demonstrated club cells’ capacity to differentiate into alveolar epithelial cells at the single-cell level. PMID:28128362

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

    PubMed

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

    2016-02-08

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

  5. Examination of the role of galectins in cell migration and re-epithelialization of wounds.

    PubMed

    Cao, Zhiyi; Saravanan, Chandrassegar; Chen, Wei-Sheng; Panjwani, Noorjahan

    2015-01-01

    Re-epithelialization is a crucial step for wound healing. As galectins play important roles in re-epithelialization, we describe here protocols for in vivo, ex vivo and in vitro examination of the role of galectins in cell migration and in re-epithelialization of wounds. For in vivo models, mouse corneas are wounded by a variety of techniques and the rate of re-epithelialization is quantified. For ex vivo organ culture models, mouse corneas are wounded in situ, the eyes are enucleated, the eyeballs are cultured in the presence or absence of galectins and the rate of re-epithelialization is quantified. For cell cultured-based in vitro assays, we examine formation of lamellipodia and activation of focal adhesion kinase in various epithelial cells.

  6. Odontogenic epithelial stem cells: hidden sources.

    PubMed

    Padma Priya, Sivan; Higuchi, Akon; Abu Fanas, Salem; Pooi Ling, Mok; Kumari Neela, Vasantha; Sunil, P M; Saraswathi, T R; Murugan, Kadarkarai; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh

    2015-12-01

    The ultimate goal of dental stem cell research is to construct a bioengineered tooth. Tooth formation occurs based on the well-organized reciprocal interaction of epithelial and mesenchymal cells. The dental mesenchymal stem cells are the best explored, but because the human odontogenic epithelium is lost after the completion of enamel formation, studies on these cells are scarce. The successful creation of a bioengineered tooth is achievable only when the odontogenic epithelium is reconstructed to produce a replica of natural enamel. This article discusses the untapped sources of odontogenic epithelial stem cells in humans, such as those present in the active dental lamina in postnatal life, in remnants of dental lamina (the gubernaculum cord), in the epithelial cell rests of Malassez, and in reduced enamel epithelium. The possible uses of these stem cells in regenerative medicine, not just for enamel formation, are discussed.

  7. Porcine Nasal Epithelial Cultures for Studies of Cystic Fibrosis Sinusitis

    PubMed Central

    Dean, Nichole; Ranganath, Neel K.; Jones, Brandon; Zhang, Shaoyan; Skinner, Daniel; Rowe, Steven M.; Sorscher, Eric J.; Woodworth, Bradford A.

    2014-01-01

    Background Transgenic cystic fibrosis (CF) murine models do not develop spontaneous lung or sinus disease, two major causes of morbidity in human CF patients. Because of these limitations, transgenic CFTR−/− pigs have been developed and are currently being characterized. These CF animal models have phenotypes closely resembling that of human CF subjects. The objectives of the current study were to develop primary porcine nasal epithelial (PNE) cultures and evaluate their usefulness as a means to investigate sinonasal transepithelial transport and CFTR function. Methods PNE derived from the septum or turbinates of CFTR+/+ and CFTR−/− pigs were cultured at an air-liquid interface to confluence and full differentiation. Epithelial monolayers were mounted in Ussing chambers to investigate pharmacologic manipulation of ion transport. Ciliary beat frequency (CBF) and scanning electron microscopy of monolayers were used to indicate degree of ciliation and cell differentiation. Results Stimulation of CFTR-mediated anion transport(ΔIsc in μA/cm2) was significantly greater in epithelia derived from the septum when compared to turbinates(33.04+/−1.17 vs. 18.9+/−0.73;p<0.05). cAMP-activated Cl− secretion was absent in CFTR−/− and present in CFTR+/+ epithelia. Calcium-activated Cl− (CaCC) secretion was increased in CF, however, overall Cl− transport through CaCCs was very low. Degree of ciliation (90%) and CBF were similar between groups. Discussion Septal PNE exhibit a robust ion transport phenotype and indicate CFTR−/− sinus disease could be attributable to diminished alternative pathways for Cl− transport. Overall, PNE have similarities to human respiratory epithelia not demonstrated in murine cells and represent useful in vitro models for studying CF sinus disease. PMID:24733748

  8. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  9. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

  10. Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods.

    PubMed

    Gildea, John J; McGrath, Helen E; Van Sciver, Robert E; Wang, Dora Bigler; Felder, Robin A

    2013-01-01

    The kidney is a highly heterogeneous organ that is responsible for fluid and electrolyte balance. Much interest is focused on determining the function of specific renal epithelial cells in humans, which can only be accomplished through the isolation and growth of nephron segment-specific epithelial cells. However, human renal epithelial cells are notoriously difficult to maintain in culture. This chapter describes the isolation, growth, immortalization, and characterization of the human renal proximal tubule cell. In addition, we describe new paradigms in 3D cell culture which allow the cells to maintain more in vivo-like morphology and function.

  11. Epithelial cell adhesion and gastrointestinal colonization of Lactobacillus in poultry.

    PubMed

    Spivey, Megan A; Dunn-Horrocks, Sadie L; Duong, Tri

    2014-11-01

    Administration of probiotic Lactobacillus cultures is an important alternative to the use of antibiotic growth promoters and has been demonstrated to improve animal health, growth performance, and preharvest food safety in poultry production. Whereas gastrointestinal colonization is thought to be critical to their probiotic functionality, factors important to Lactobacillus colonization in chickens are not well understood. In this study we investigate epithelial cell adhesion in vitro and colonization of Lactobacillusin vivo in broiler chickens. Adhesion of Lactobacillus cultures to epithelial cells was evaluated using the chicken LMH cell line. Lactobacillus cultures were able adhere effectively to LMH cells relative to Bacillus subtilis and Salmonella Typhimurium. Epithelial cell adhesion was similar for Lactobacillus crispatus TDCC 75, L. cristpatus TDCC 76, and Lactobacillus gallinarum TDCC 77, and all 3 were more adherent than L. gallinarum TDCC 78. However, when colonization was evaluated in the ileum and cecum of broiler chicks, L. crispatus TDCC 75 and L. gallinarum TDCC 77 were more persistent than L. crispatus TDCC 76 and L. gallinarum TDCC 78. The reduction of growth in medium supplemented with oxgal was greater for L. gallinarum TDCC 78 than L. gallinarum TDCC 77, suggesting that whereas adhesion was similar for the 2 strains, the difference in colonization between L. gallinarum strains may be due in part to their bile sensitivity. This study demonstrates that whereas adhesion to epithelial cells may be important in predicting gastrointestinal colonization, other factors including bile tolerance may also contribute to the colonization of Lactobacillus in poultry. Additionally, the chicken LMH cell line is expected to provide a platform for investigating mechanisms of Lactobacillus adhesion to epithelial tissue and evaluating the probiotic potential Lactobacillus in poultry.

  12. A Common Stem Cell for Murine Cortical and Medullary Thymic Epithelial Cells?

    PubMed Central

    Van Soest, Peter; Platenburg, Peter Paul; Van Ewijk, Willem

    1995-01-01

    We have addressed the question whether the epithelial stroma in the thymus is derived from a common stem cell or whether cortical and medullary epithelial cells are derived from different embryonic stem cells emerging, for example, from endoderm and ectoderm. By the use of rapidly expanding cultures of thymic epithelial cells (TEC) from 14 to 16 day-old murine fetuses and by specific antibodies against cortical and medullary epithelium, respectively, we were able to demonstrate a small subpopulation of double-labeled TEC in the cultures. These cells were not present in TEC cultures initiated from thymuses of neonatal mice. Double-labeled TEC were also found in tissue sections from fetal thymuses. These findings may indicate that TEC populations of the cortex and the medulla are derived from a common stem cell, with potential for differentiation toward both cortical and medullary TEC. PMID:9700364

  13. Directed differentiation of airway epithelial cells of human bone marrow mesenchymal stem cells.

    PubMed

    Li, Jian-Dong

    2016-11-01

    The ability to generate lung and airway epithelial cells from human bone marrow mesenchymal stem cells (hBMSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening, and studies of human lung development. In this research, hBMSCs were cultured in specialized airway epithelial cell growth media for differentiation of airway epithelial cells, including keratinocyte growth factor transferrin, bovine pituitary extract, epinephrine, triiodothyronine and retinoic acid. The surfactant protein C, a specific marker of type II pneumocytes, and its corresponding protein were demonstrated by immunofluorescence and western blotting after differentiation of airway epithelial cells, respectively. These cells were then transferred into an induced acute lung injury model. The results showed that the hBMSCs could induce differentiation in airway epithelial cells under the special conditions of the medium, the result for surfactant protein C was positive in differentiated airway epithelial cells using immunofluorescence and western blotting, and these cells were successfully colonized in the injured lung airway. In conclusion, our research shows that a population of airway epithelial cells can be specifically generated from hBMSCs and that induced cells may be allowed to participate in tissue repair.

  14. Contact inhibition of phagocytosis in epithelial sheets: alterations of cell surface properties induced by cell-cell contacts.

    PubMed

    Vasiliev, J M; Gelfand, I M; Domnina, L V; Zacharova, O S; Ljubimov, A V

    1975-02-01

    Contact inhibition of phagocytosis was found to be characteristic for epithelial sheets formed in cultures by several cell types: normal and transformed mouse kidney cells, and differentiated mouse hepatoma cells. In these sheets most central cells surrounded by other cells had very low phagocytic activity. In contrast, marginal cells having a free edge were able to perform an active phagocytosis. Contact inhibition of phagocytosis was absent in dense cultures of mouse embryo fibroblasts and in cultures of anaplastic mouse hepatoma 22a. The upper surface of epithelial sheets was nonadhesive for prelabeled epithelial cells and fibroblasts. In contrast, the upper surface of dense cultures of mouse fibroblasts was adhesive for these cells. These and other data strengthen the suggestion that contact inhibition of phagocytosis is a result of different adhesiveness of the upper cell surface and of the surfaces near the free edge. Agents inhibiting cell surface movements at the free edges of marginal epithelial cells (cytochalasin, azide, sorbitol, low temperature) prevented adhesion of particles to these edges. Possibly, the surface of actively moving cytoplasmic processes is the only cell part that has adhesive properties necessary for the formation of attachments with other cellular and noncellular surfaces. In epithelial sheets, in contrast to fibroblast cultures, Colcemid did not activate movements of immobile contacting cell edges. These results indicate that mechanisms of contact immobilization of cell surface may be different in epithelium and fibroblasts. Firm contacts formed between epithelial cells are sufficient for stable immobilization of the surface; additional stabilization of the surface by microtubules is not essential. Fibroblasts do not form firm contacts and the Colcemid-sensitive stabilization process is essential for maintenance of the immobile state of their surfaces. Differences in the stability of cell surface immobilization produced by cell-cell

  15. Modelling the effects of microgravity on the permeability of air interface respiratory epithelial cell layers

    NASA Astrophysics Data System (ADS)

    dos Santos, Marlise A.; Bosquillon, Cynthia; Russomano, Thais; Sundaresan, Alamelu; Falcão, Felipe; Marriott, Christopher; Forbes, Ben

    2010-09-01

    Although it has been suggested that microgravity might affect drug absorption in vivo, drug permeability across epithelial barriers has not yet been investigated in vitro during modelled microgravity. Therefore, a cell culture/diffusion chamber was designed specifically to accommodate epithelial cell layers in a 3D-clinostat and allow epithelial permeability to be measured under microgravity conditions in vitro with minimum alteration to established cell culture techniques. Human respiratory epithelial Calu-3 cell layers were used to model the airway epithelium. Cells grown at an air interface in the diffusion chamber from day 1 or day 5 after seeding on 24-well polyester Transwell cell culture inserts developed a similar transepithelial electrical resistance (TER) to cells cultured in conventional cell culture plates. Confluent Calu-3 layers exposed to modelled microgravity in the 3D-clinostat for up to 48 h maintained their high TER. The permeability of the paracellular marker 14C-mannitol was unaffected after a 24 h rotation of the cell layers in the 3D-clinostat, but was increased 2-fold after 48 h of modelled microgravity. It was demonstrated that the culture/diffusion chamber developed is suitable for culturing epithelial cell layers and, when subjected to rotation in the 3D-clinostat, will be a valuable in vitro system in which to study the influence of microgravity on epithelial permeability and drug transport.

  16. Evidence for epithelial-mesenchymal transitions in adult liver cells.

    PubMed

    Sicklick, Jason K; Choi, Steve S; Bustamante, Marcia; McCall, Shannon J; Pérez, Elizabeth Hernández; Huang, Jiawen; Li, Yin-Xiong; Rojkind, Marcos; Diehl, Anna Mae

    2006-10-01

    Both myofibroblastic hepatic stellate cells (HSC) and hepatic epithelial progenitors accumulate in damaged livers. In some injured organs, the ability to distinguish between fibroblastic and epithelial cells is sometimes difficult because cells undergo epithelial-mesenchymal transitions (EMT). During EMT, cells coexpress epithelial and mesenchymal cell markers. To determine whether EMT occurs in adult liver cells, we analyzed the expression profile of primary HSC, two HSC lines, and hepatic epithelial progenitors. As expected, all HSC expressed HSC markers. Surprisingly, these markers were also expressed by epithelial progenitors. In addition, one HSC line expressed typical epithelial progenitor mRNAs, and these epithelial markers were inducible in the second HSC line. In normal and damaged livers, small ductular-type cells stained positive for an HSC marker. In conclusion, HSC and hepatic epithelial progenitors both coexpress epithelial and mesenchymal markers, providing evidence that EMT occurs in adult liver cells.

  17. Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera.

    PubMed Central

    Asch, B B; Burstein, N A; Vidrich, A; Sun, T T

    1981-01-01

    Few markers are available to identify the three types of mammary epithelial cells--ductal, alveolar, and myoepithelial--especially in pathological conditions and in cell cultures. We have used antisera to human keratins in immunofluorescence to facilitate the identification of the three mouse mammary epithelial cell types. In frozen tissue sections and primary cell cultures, a rabbit antikeratin antiserum specifically stained cytoplasmic filaments in all three types of epithelial cells. A guinea pig antiserum against the same keratin preparation, however, reacted preferentially with filaments in myoepithelial cells and readily detected this cell type in normal, dysplastic, and malignant mammary tissues and cell cultures. Neither antisera reacted with fibroblasts or any other mesenchymal cells. The combined use of the two antikeratin antisera thereby permits rapid surveys of tissue sections and cultures for the localization of not only all epithelial cells but also the subpopulation of myoepithelial cells. Moreover, when mammary cultures established from late-pregnant or lactating mice were stained simultaneously with guinea pig antikeratin and rabbit anticasein antisera, three populations of epithelial cells were mutually exclusive: those stained by anticasein antiserum, those stained by guinea pig antikeratin antiserum, and those stained by neither, consistent with properties of alveolar, myoepithelial, and ductal cells, respectively. These antisera thus offer a tool for studying different epithelial cell types during mammary development, tumorigenesis, and malignant progression. Images PMID:6170984

  18. Epithelial stem cells and intestinal cancer.

    PubMed

    Tan, Shawna; Barker, Nick

    2015-06-01

    The mammalian intestine is comprised of an epithelial layer that serves multiple functions in order to maintain digestive activity as well as intestinal homeostasis. This epithelial layer contains highly proliferative stem cells which facilitate its characteristic rapid regeneration. How these stem cells contribute to tissue repair and normal homeostasis are actively studied, and while we have a greater understanding of the molecular mechanisms and cellular locations that underlie stem cell regulation in this tissue, much still remains undiscovered. This review describes epithelial stem cells in both intestinal and non-intestinal tissues, as well as the strategies that have been used to further characterize the cells. Through a discussion of the current understanding of intestinal self-renewal and tissue regeneration in response to injury, we focus on how dysregulation of critical signaling pathways results in potentially oncogenic aberrations, and highlight issues that should be addressed in order for effective intestinal cancer therapies to be devised.

  19. Epithelial cell extrusion: Pathways and pathologies.

    PubMed

    Gudipaty, Swapna Aravind; Rosenblatt, Jody

    2016-05-19

    To remove dying or unwanted cells from an epithelium while preserving the barrier function of the layer, epithelia use a unique process called cell extrusion. To extrude, the cell fated to die emits the lipid Sphingosine 1 Phosphate (S1P), which binds the G-protein-coupled receptor Sphingosine 1 Phosphate receptor 2 (S1P2) in the neighboring cells that activates Rho-mediated contraction of an actomyosin ring circumferentially and basally. This contraction acts to squeeze the cell out apically while drawing together neighboring cells and preventing any gaps to the epithelial barrier. Epithelia can extrude out cells targeted to die by apoptotic stimuli to repair the barrier in the face of death or extrude live cells to promote cell death when epithelial cells become too crowded. Indeed, because epithelial cells naturally turn over by cell death and division at some of the highest rates in the body, epithelia depend on crowding-induced live cell extrusion to preserve constant cell numbers. If extrusion is defective, epithelial cells rapidly lose contact inhibition and form masses. Additionally, because epithelia act as the first line of defense in innate immunity, preservation of this barrier is critical for preventing pathogens from invading the body. Given its role in controlling constant cell numbers and maintaining barrier function, a number of different pathologies can result when extrusion is disrupted. Here, we review mechanisms and signaling pathways that control epithelial extrusion and discuss how defects in these mechanisms can lead to multiple diseases. We also discuss tactics pathogens have devised to hijack the extrusion process to infect and colonize epithelia.

  20. Gallic acid ameliorates renal functions by inhibiting the activation of p38 MAPK in experimentally induced type 2 diabetic rats and cultured rat proximal tubular epithelial cells.

    PubMed

    Ahad, Amjid; Ahsan, Haseeb; Mujeeb, Mohd; Siddiqui, Waseem Ahmad

    2015-10-05

    Diabetic nephropathy (DN) is one of the leading causes of morbidity and mortality in diabetic patients that accounts for about 40% of deaths in type 2 diabetes. p38 mitogen activated protein kinase (p38 MAPK), a serine-threonine kinase, plays an important role in tissue inflammation and is known to be activated under conditions of oxidative stress and hyperglycemia. The role of p38 MAPK has been demonstrated in DN, and its inhibition has been suggested as an alternative approach in the treatment of DN. In the present study, we investigated the nephroprotective effects of an anti-inflammatory phenolic compound, gallic acid (GA, 3,4,5-trihydroxybenzoic acid), in high fat diet/streptozotocin (HFD/STZ) induce type 2 diabetic wistar albino rats. GA (25 mg/kgbw and 50 mg/kgbw, p.o.) treatment for 16 weeks post induction of diabetes led to a significant reduction in the levels of blood glucose, HbA1c, serum creatinine, blood urea nitrogen and proteinuria as well as a significant reduction in the levels of creatinine clearance. GA significantly inhibited the renal p38 MAPK and nuclear factor kappa B (N-κB) activation as well as significantly reduced the levels of renal transforming growth factor beta (TGF-β) and fibronectin. Treatment with GA resulted in a significant reduction in the serum levels of proinflammatory cytokines viz. interleukin 1 beta (IL-1β), IL-6 and tumor necrosis factor alpha (TNF-α). Moreover, GA significantly lowered renal pathology and attenuated renal oxidative stress. In cultured rat NRK 52E proximal tubular epithelial cells, GA treatment inhibited high glucose induced activation of p38 MAPK and NF-κB as well as suppressed proinflammatory cytokine synthesis. The results of the present study provide in vivo and in vitro evidences that the p38 MAPK pathway plays an important role in the pathogenesis of DN, and GA attenuates the p38 MAPK-mediated renal dysfunction in HFD/STZ induced type 2 diabetic rats.

  1. Macrophages promote benzopyrene-induced tumor transformation of human bronchial epithelial cells by activation of NF-κB and STAT3 signaling in a bionic airway chip culture and in animal models.

    PubMed

    Li, Encheng; Xu, Zhiyun; Zhao, Hui; Sun, Zhao; Wang, Lei; Guo, Zhe; Zhao, Yang; Gao, Zhancheng; Wang, Qi

    2015-04-20

    We investigated the role of macrophages in promoting benzopyrene (BaP)-induced malignant transformation of human bronchial epithelial cells using a BaP-induced tumor transformation model with a bionic airway chip in vitro and in animal models. The bionic airway chip culture data showed that macrophages promoted BaP-induced malignant transformation of human bronchial epithelial cells, which was mediated by nuclear factor (NF)-κB and STAT3 pathways to induce cell proliferation, colony formation in chip culture, and tumorigenicity in nude mice. Blockage of interleukin (IL)-6 or tumor necrosis factor (TNF)-α signaling or inhibition of NF-κB, STAT3, or cyclinD1 expression abrogated the effect of macrophages on malignant transformation in the bionic airway chip culture. In vivo, macrophages promoted lung tumorigenesis in a carcinogen-induced animal model. Similarly, blockage of NF-κB, STAT3, or cyclinD1 using siRNA transfection decreased the carcinogen-induced tumorigenesis in rats. We demonstrated that macrophages are critical in promoting lung tumorigenesis and that the macrophage-initiated TNF-α/NF-κB/cyclinD1 and IL-6/STAT3/cyclinD1 pathways are primarily responsible for promoting lung tumorigenesis.

  2. Macrophages promote benzopyrene-induced tumor transformation of human bronchial epithelial cells by activation of NF-κB and STAT3 signaling in a bionic airway chip culture and in animal models

    PubMed Central

    Sun, Zhao; Wang, Lei; Guo, Zhe; Zhao, Yang; Gao, Zhancheng; Wang, Qi

    2015-01-01

    We investigated the role of macrophages in promoting benzopyrene (BaP)-induced malignant transformation of human bronchial epithelial cells using a BaP-induced tumor transformation model with a bionic airway chip in vitro and in animal models. The bionic airway chip culture data showed that macrophages promoted BaP-induced malignant transformation of human bronchial epithelial cells, which was mediated by nuclear factor (NF)-κB and STAT3 pathways to induce cell proliferation, colony formation in chip culture, and tumorigenicity in nude mice. Blockage of interleukin (IL)-6 or tumor necrosis factor (TNF)-α signaling or inhibition of NF-κB, STAT3, or cyclinD1 expression abrogated the effect of macrophages on malignant transformation in the bionic airway chip culture. In vivo, macrophages promoted lung tumorigenesis in a carcinogen-induced animal model. Similarly, blockage of NF-κB, STAT3, or cyclinD1 using siRNA transfection decreased the carcinogen-induced tumorigenesis in rats. We demonstrated that macrophages are critical in promoting lung tumorigenesis and that the macrophage-initiated TNF-α/NF-κB/cyclinD1 and IL-6/STAT3/cyclinD1 pathways are primarily responsible for promoting lung tumorigenesis. PMID:25823926

  3. Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells.

    PubMed

    Nagahara, Teruya; Shiraha, Hidenori; Sawahara, Hiroaki; Uchida, Daisuke; Takeuchi, Yasuto; Iwamuro, Masaya; Kataoka, Junro; Horiguchi, Shigeru; Kuwaki, Takeshi; Onishi, Hideki; Nakamura, Shinichiro; Takaki, Akinobu; Nouso, Kazuhiro; Yamamoto, Kazuhide

    2015-09-01

    Microenvironment plays an important role in epithelial-mesenchymal transition (EMT) and stemness of cells in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) is known as a tumor stemness marker of HCC. To investigate the relationship between microenvironment and stemness, we performed an in vitro co-culture assay. Four HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) were co-cultured with the TWNT-1 immortalized hepatic stellate cells (HSCs), which create a microenvironment with HCC. Cell proliferation ability was analyzed by flow cytometry (FCM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while migration ability was assessed by a wound healing assay. Expression of EpCAM was analyzed by immunoblotting and FCM. HCC cell lines were co-cultured with TWNT-1 treated with small interfering RNA (siRNA) for TGF-β and HB-EGF; we then analyzed proliferation, migration ability and protein expression using the methods described above. Proliferation ability was unchanged in HCC cell lines co-cultured with TWNT-1. Migration ability was increased in HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) directly (216.2±67.0, 61.0±22.0, 124.0±66.2 and 51.5±40.3%) and indirectly (102.5±22.0, 84.6±30.9, 86.1±25.7 and 73.9±29.7%) co-cultured with TWNT-1 compared with the HCC uni-culture. Immunoblot analysis revealed increased EpCAM expression in the HCC cell lines co-cultured with TWNT-1. Flow cytometry revealed that the population of E-cadherin-/N-cadherin+ and EpCAM-positive cells increased and accordingly, EMT and stemness in the HCC cell line were activated. These results were similar in the directly and indirectly co-cultured samples, indicating that humoral factors were at play. Conversely, HCC cell lines co-cultured with siRNA‑treated TWNT-1 showed decreased migration ability, a decreased population of EpCAM-positive and E-cadherin-/N-cadherin+ cells. Taken together, humoral factors secreted from TWNT-1

  4. The Three-Dimensional Culture of Epithelial Organoids Derived from Embryonic Chicken Intestine.

    PubMed

    Pierzchalska, Malgorzata; Panek, Malgorzata; Czyrnek, Malgorzata; Grabacka, Maja

    2016-10-28

    The intestinal epithelium isolated from chicken embryos in last 3 days of development can be used to establish the 3D culture of intestinal organoids. When fragments of epithelial tissue released by incubation with EGTA (2.5 mM, 2 h) are embedded in Matrigel matrix on cell culture inserts the formation of empty spheres covered by epithelial cells is observed in first 24 h of culture. The growth and survival of organoids are supported by the addition of R-spondin 1, Noggin, and prostaglandin E2 to the culture medium. The organoids are accompanied by myofibroblasts which become visible in the next 2 days of culture. The intestinal enteroids (free of myofibroblasts) can be obtained from adult chicken intestine.

  5. Advances in cell culture

    SciTech Connect

    Maramorosch, K. )

    1987-01-01

    This book presents papers on advances in cell culture. Topics covered include: Genetic changes in the influenza viruses during growth in cultured cells; The biochemistry and genetics of mosquito cells in culture; and Tree tissue culture applications.

  6. Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

    SciTech Connect

    Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M.; Mondal, Sumona; Woodworth, Craig D.

    2012-03-30

    The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.

  7. Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization.

    PubMed

    Vandermark, Erik R; Deluca, Krysta A; Gardner, Courtney R; Marker, Daniel F; Schreiner, Cynthia N; Strickland, David A; Wilton, Katelynn M; Mondal, Sumona; Woodworth, Craig D

    2012-03-30

    The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.

  8. Oxidized alginate hydrogels as niche environments for corneal epithelial cells.

    PubMed

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-10-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2-0.8 µm) than unmodified gels (pore diameter: 0.05-0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

  9. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    PubMed Central

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  10. Long-term homeostasis and wound healing in an in vitro epithelial stem cell niche model

    PubMed Central

    Miyashita, Hideyuki; Niwano, Hiroko; Yoshida, Satoru; Hatou, Shin; Inagaki, Emi; Tsubota, Kazuo; Shimmura, Shigeto

    2017-01-01

    Cultures of epithelial cells are limited by the proliferative capacity of primary cells and cell senescence. Herein we show that primary human epithelial cell sheets cultured without dermal equivalents maintained homeostasis in vitro for at least 1 year. Transparency of these sheets enabled live observation of pigmented melanocytes and Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) labeled epithelial cells during wound healing. Cell turn over and KRT15 expression pattern stabilized within 3 months, when KRT15 bright clusters often associated with niche-like melanocytes became apparent. EdU labels were retained in a subset of epithelial cells and melanocytes after 6 months chasing, suggesting their slow cell cycling property. FUCCI-labeling demonstrated robust cell migration and proliferation following wounding. Transparency and long-term (1 year) homeostasis of this model will be a powerful tool for the study of wound healing and cell linage tracing. PMID:28233843

  11. Keratin cytoskeletons in epithelial cells of internal organs

    PubMed Central

    Sun, Tung-Tien; Shih, Chiaho; Green, Howard

    1979-01-01

    An antiserum against human epidermal keratins was used to detect keratins in frozen sections of various rabbit and human tissues by indirect immunofluorescence. Strong staining was observed in all stratified squamous epithelia (epidermis, cornea, conjunctiva, tongue, esophagus, vagina, and anus), in epidermal appendages (hair follicle, sebaceous gland, ductal and myoepithelial cells of sweat glands), as well as in Hassall's corpuscles of the thymus, indicating that all contain abundant keratins. No staining by the antiserum was observed in fibroblasts, muscle of any type, cartilage, blood vessel, nerve tissue, iris or lens epithelium, or the glomerular or tubular cells of the kidney. In contrast, the antiserum stained the cells of most epithelia of the intestinal tract, urinary tract (urethra, bladder, ureter, collecting ducts of kidney), female genital tract (cervix, cervical glands, uterus, and oviduct), and respiratory tract (trachea and bronchi). Epithelial cells of the fine ductal system in the pancreas and submaxillary gland also stained well. When primary cultures of epithelial cells derived from bladder, intestine, kidney, and trachea were grown on glass coverslips and stained with anti-keratin, fiber networks similar to those of cultured keratinocytes were observed. These results show that keratins constitute a cytoskeleton in epithelial cells of diverse morphology and embryological origin. The stability of keratin filaments probably confers the structural strength necessary for cells covering a free surface. Keratin staining can be used to obtain information about the origin of cell lines. Images PMID:111242

  12. Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells

    PubMed Central

    Sajjan, Umadevi; Wang, Qiong; Zhao, Ying; Gruenert, Dieter C.; Hershenson, Marc B.

    2008-01-01

    Rationale: Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. Objectives: We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. Methods: Primary human airway epithelial cells grown at air–liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (RT) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. Measurements and Main Results: Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in RT without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease RT, suggesting a requirement for viral replication. Reduced RT was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-α, IFN-γ and IL-1β reversed corresponding cytokine-induced reductions in RT but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. Conclusions: RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function. PMID:18787220

  13. Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration.

    PubMed

    Coraux, Christelle; Roux, Jacqueline; Jolly, Thomas; Birembaut, Philippe

    2008-08-15

    In healthy subjects, the respiratory epithelium forms a continuous lining to the airways and to the environment, and plays a unique role as a barrier against external deleterious agents to protect the airways from the insults. In respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), chronic bronchitis, or asthma, the airway epithelium is frequently remodeled and injured, leading to the impairment of its defense functions. The rapid restoration of the epithelial barrier is crucial for these patients. The complete regeneration of the airway epithelium is a complex phenomenon, including not only the epithelial wound repair but also the epithelial differentiation to reconstitute a fully well differentiated and functional epithelium. The regeneration implies two partners: the epithelial stem/progenitor cells and factors able to regulate this process. Among these factors, epithelial cells-extracellular matrix (ECM) interactions play a crucial role. The secretion of a provisional ECM, the cell-ECM relationships through epithelial receptors, and the remodeling of the ECM by proteases (mainly matrix metalloproteinases) contribute not only to airway epithelial repair by modulating epithelial cell migration and proliferation, but also to the differentiation of repairing cells leading to the complete restoration of the wounded epithelium. A better characterization of resident stem cells and of effectors of the regeneration process is an essential prerequisite to propose new regenerative therapeutics to patients suffering from infectious/inflammatory respiratory diseases.

  14. Induction of pyrimidine dimers and unscheduled DNA synthesis in cultured mouse epithelial cells exposed to 254-nm- and u. v. -B radiation

    SciTech Connect

    Yotti, L.P.; Ley, R.D.

    1983-01-01

    The induction and fate of pyrimidine dimers and unscheduled DNA synthesis were measured in u.v.-irradiated primary, newborn SENCAR mouse epithelial cells. Unscheduled DNA synthesis was induced in a dose responsive manner by two u.v. sources, a germicidal lamp (254 nm) and an FS40 sunlamp (280--400 nm). Using the endonuclease-sensitive site assay to detect pyrimidine dimer production and excision, we examined the response of the newborn mouse cells to both u.v. sources. We were unable to detect the removal of pyrimidine dimers with either of the two sources of u.v. The speculation is made that primary, newborn mouse epidermal cells excise u.v.-induced pyrimidine dimers to an extent below the level of detection of the endonuclease-sensitive site assay but to an extent sufficient to induce unscheduled DNA synthesis.

  15. Gradient isolation of glial cells: evidence that flat epithelial cells are astroglial cell precursors.

    PubMed

    Meller, K

    1987-07-01

    Discontinuous gradients of metrizamide were used to separate the cell components of monolayers of primary cultures of embryonic rat brains. These primary cell cultures were of two types: long-term cultures (more than a year) of embryonic rat brain, which contained several glial cell types, and monolayers of cell cultures (several weeks old), which contained a complex population of cells, including neuronal elements. The gradient separation produces fractions of pure flat epithelial cells that are able to survive and proliferate. After a few days, all flat epithelial cells become confluent and show a positive reaction to glial fibrillary acidic protein (GFAP); this indicates that these cells astroglial precursor cells. Following their maintenance in vitro for several months, all cultures give rise to a pure population of astrocytes identified not only by their characteristic morphology, but also by their content of GFAP. It is proposed that the differentiation controls are dependent on cell interactions that are influenced by the composition of the cell population and/or the molecular growth and differentiation factors released by these cells into the medium.

  16. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

    PubMed

    Lindemans, Caroline A; Calafiore, Marco; Mertelsmann, Anna M; O'Connor, Margaret H; Dudakov, Jarrod A; Jenq, Robert R; Velardi, Enrico; Young, Lauren F; Smith, Odette M; Lawrence, Gillian; Ivanov, Juliet A; Fu, Ya-Yuan; Takashima, Shuichiro; Hua, Guoqiang; Martin, Maria L; O'Rourke, Kevin P; Lo, Yuan-Hung; Mokry, Michal; Romera-Hernandez, Monica; Cupedo, Tom; Dow, Lukas E; Nieuwenhuis, Edward E; Shroyer, Noah F; Liu, Chen; Kolesnick, Richard; van den Brink, Marcel R M; Hanash, Alan M

    2015-12-24

    Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.

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

    EPA Science Inventory

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

  18. Detonation Nanodiamond Toxicity in Human Airway Epithelial Cells Is Modulated by Air Oxidation

    EPA Science Inventory

    Detonational nanodiamonds (DND), a nanomaterial with an increasing range of industrial and biomedical applications, have previously been shown to induce a pro-inflammatory response in cultured human airway epithelial cells (HAEC). We now show that surface modifications induced by...

  19. Cl- secretion induced by bile salts. A study of the mechanism of action based on a cultured colonic epithelial cell line.

    PubMed Central

    Dharmsathaphorn, K; Huott, P A; Vongkovit, P; Beuerlein, G; Pandol, S J; Ammon, H V

    1989-01-01

    When applied to the basolateral (serosal) side of the T84 colonic epithelial monolayer, taurodeoxycholate caused net Cl- secretion in a dose-dependent manner with a threshold effect observed at 0.2 mM. In contrast, when applied to the apical (luminal) surface, concentrations of taurodeoxycholate below 1 mM had little or no effect. Only when the concentration of taurodeoxycholate present on the apical side was greater than or equal to 1 mM did apical addition results in an electrolyte transport effect. This apical effect on electrolyte transport was associated with an abrupt increase in the permeability of the monolayer. Cyclic AMP and cyclic GMP in the T84 monolayers were not increased by the bile salt, but in the presence of extracellular Ca2+, free cytosolic Ca2+ increased with a graded dose effect and time course that corresponded approximately to the changes in short circuit current (Isc). The results suggest that luminal bile salts at a relatively high concentration (greater than or equal to 1 mM) increase tight junction permeability. Once tight junction permeability increases, luminal bile salts could reach the basolateral membrane of the epithelial cells where they act to increase free cytosolic Ca2+ from extracellular sources. The resulting increases in free cytosolic Ca2+, rather than in cyclic nucleotides, appear to be involved in transcellular Cl- secretion. PMID:2547841

  20. In vitro isolation and cultivation of rabbit tracheal epithelial cells using tissue explant technique.

    PubMed

    Shi, Hong-Can; Lu, Dan; Li, Hai-Jia; Han, Shi; Zeng, Yan-Jun

    2013-04-01

    Epithelial cells from tracheal mucosa offer significant potential as a cell source in development of tissue-engineered trachea. The purpose of this study was to investigate and optimize a suitable culture system for tracheal epithelial cells, including the methods of primary culture, passage, identification, and cryopreservation. Epithelial cells were isolated from rabbit tracheal mucosa using tissue explant technique and were subjected to immunohistochemistry, immunofluorescence, and cryopreservation after purification. Epithelial cells reached confluency at 14-15 d. Immunohistochemical staining for cytokeratin showed brown yellow-positive cytoplasm and blue-counterstained nuclei, while immunofluorescence staining for cytokeratin showed green-positive cytoplasm and clear cell outline, indicating that the cultured cells had properties of epithelial cells. After recovery, epithelial cells exhibited high survival and viability. The results demonstrated that in vitro isolation and cultivation model was successfully established to provide high proliferative capacity, typical morphology and characteristics of tracheal epithelial cells from trachea mucosa by the use of the tissue explant technique.

  1. Expression of inducible nitric oxide in human lung epithelial cells.

    PubMed

    Robbins, R A; Barnes, P J; Springall, D R; Warren, J B; Kwon, O J; Buttery, L D; Wilson, A J; Geller, D A; Polak, J M

    1994-08-30

    Nitric oxide (NO) is increased in the exhaled air of subjects with several airway disorders. To determine if cytokines could stimulate epithelial cells accounting for the increased NO, the capacity of the proinflammatory cytokines (cytomix: tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma) to increase inducible nitric oxide synthase (iNOS) was investigated in A549 and primary cultures of human bronchial epithelial cells. Cytomix induced a time-dependent increase in nitrite levels in culture supernatant fluids (p < 0.05). Increased numbers of cells stained for iNOS and increased iNOS mRNA was detected in the cytokine-stimulated cells compared to control (p < 0.05). Dexamethasone diminished the cytokine-induced increase in nitrite, iNOS by immunocytochemistry, and iNOS mRNA. These data demonstrate that cytokines, such as those released by mononuclear cells, can induce lung epithelial iNOS expression and NO release, and that this is attenuated by dexamethasone.

  2. Human eccrine sweat gland epithelial cultures express ductal characteristics.

    PubMed Central

    Brayden, D J; Cuthbert, A W; Lee, C M

    1988-01-01

    1. Isolated human eccrine sweat glands were cultured in vitro. Cells were harvested and plated onto permeable supports to form confluent cell sheets, area 0.2 cm2. These were used to study the electrogenic transepithelial transport of ions by measurement of short-circuit current (SCC). Epithelial sheets had a basal SCC of 5.89 +/- 0.62 microA cm-2 (n = 33) and a transepithelial resistance of 74.1 +/- 5.6 omega cm2 (n = 33). The transepithelial potential difference varied between -0.2 and -1.8 mV with a mean value of -0.71 +/- 0.09 mV (n = 33). 2. The basal current was abolished by addition of 10 microM-amiloride to the apical bathing solution. The concentration of amiloride which inhibited basal SCC by 50% (EC50) was 0.4 microM. Cultures prepared from the secretory coil of sweat glands, rather than from whole glands, were similarly sensitive to amiloride (EC50 = 0.8 microM). 3. Lysylbradykinin (LBK), carbachol, isoprenaline, prostaglandin E2 (PGE2) and A23187 all increased SCC in cultures from whole glands. LBK responses were obtained with basolateral and not with apical application. Furthermore LBK actions were not substantially altered by cyclo-oxygenase inhibition but showed marked desensitization upon repeated application. Sheet cultures prepared from sweat gland coils also showed SCC responses to both carbachol and LBK. Forskolin, an activator of adenylate cyclase, did not alter SCC in either type of preparation. 4. Replacement of chloride and of chloride and bicarbonate in the bathing solution did not cause attenuation of the responses to LBK or carbachol in whole-gland sheet cultures. Furthermore responses were unaffected by piretanide or acetazolamide. These results were taken to indicate that anion secretion was not the basis for the SCC responses. 5. Responses to LBK and carbachol were significantly reduced by amiloride (10 microM), this effect being reversible. No responses to LBK or carbachol were seen when N-methyl-D-glucamine (NMDG) was used to

  3. Requirements for invasion of epithelial cells by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Sreenivasan, P K; Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Actinobacillus actinomycetemcomitans, an oral bacterium implicated in human periodontal disease, was recently demonstrated to invade cultured epithelial cells (D. H. Meyer, P. K. Sreenivasan, and P. M. Fives-Taylor, Infect. Immun. 59:2719-2726, 1991). This report characterizes the requirements for invasion of KB cells by A. actinomycetemcomitans. The roles of bacterial and host factors were investigated by using selective agents that influence specific bacterial or host cell functions. Inhibition of bacterial protein synthesis decreased invasion, suggesting the absence of a preformed pool of proteins involved in A. actinomycetemcomitans invasion. Inhibition of bacterial and eukaryotic energy synthesis also decreased invasion, confirming that A. actinomycetemcomitans invasion is an active process. Bacterial adherence to KB cells was indicated by scanning electron microscopy of infected KB cells. Further, the addition of A. actinomycetemcomitans-specific serum to the bacterial inoculum reduced invasion substantially, suggesting a role for bacterial attachment in invasion. Many of the adherent bacteria invaded the epithelial cells under optimal conditions. Inhibitors of receptor-mediated endocytosis inhibited invasion by A. actinomycetemcomitans. Like that of many facultatively intracellular bacteria, A. actinomycetemcomitans invasion was not affected by eukaryotic endosomal acidification. These are the first published observations describing the requirements for epithelial cell invasion by a periodontopathogen. They demonstrate that A. actinomycetemcomitans utilizes a mechanism similar to those used by many but not all invasive bacteria to gain entry into eukaryotic cells. Images PMID:8454326

  4. Basolateral Cl channels in primary airway epithelial cultures.

    PubMed

    Fischer, Horst; Illek, Beate; Finkbeiner, Walter E; Widdicombe, Jonathan H

    2007-06-01

    Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.

  5. Suitable in vitro culture of Eimeria bovis meront II stages in bovine colonic epithelial cells and parasite-induced upregulation of CXCL10 and GM-CSF gene transcription.

    PubMed

    Hermosilla, Carlos; Stamm, Ivonne; Menge, Christian; Taubert, Anja

    2015-08-01

    We here established a suitable in vitro cell culture system based on bovine colonic epithelial cells (BCEC) for the development of Eimeria bovis merozoites I and the characterization of early parasite-induced innate epithelial host cell reactions as gene transcription of proinflammatory molecules. Both primary and permanent BCEC (BCEC (rim) and BCEC(perm)) were suitable for E. bovis merozoite I invasion and subsequent development of meronts II leading to the release of viable merozoites II. E. bovis merozoite II failed to develop any further neither into gamont nor oocyst stages in BCEC in vitro. E. bovis merozoite I induced innate epithelial host cell reactions at the level of CXC/CCL chemokines (CXCL1, CXCL8, CXCL10, CCL2), IL-6, and GM-CSF gene transcription. Overall, both BCEC types were activated by merozoite I infections since they showed significantly enhanced gene transcript levels of the immunomodulatory molecules CXCL10 and GM-CSF. However, gene transcription profiles of BCEC(prim) and BCEC(perm) revealed different reaction patterns in response to merozoite I infection with regard to quality and kinetics of chemokine/cytokine gene transcription. Although both BCEC types equally showed most prominent responses for CXCL10 and GM-CSF, the induction of CXCL1, CXCL8, CCL2, and IL-6 gene transcripts varied qualitatively and quantitatively. Our results demonstrate that BCEC seem capable to respond to E. bovis merozoite I infection by the upregulation of CXCL10 and GM-CSF gene transcription and therefore probably contribute to host innate effector mechanisms against E. bovis.

  6. CCN1 induces a reversible epithelial-mesenchymal transition in gastric epithelial cells.

    PubMed

    Chai, Jianyuan; Norng, Manith; Modak, Cristina; Reavis, Kevin M; Mouazzen, Wasim; Pham, Jennifer

    2010-08-01

    CCN1 is a matricellular protein that activates many genes related to wound healing and tissue remodeling in fibroblasts, but its effect on epithelial cells remains unclear. This study examined the role of CCN1 in epithelial wound healing using rat gastric epithelial cells and rat stomach ulcer as in vitro and in vivo models, respectively. We found that CCN1 expression is highly upregulated in the epithelial cells adjacent to a wound and remains high until the wound is healed. Upregulation of CCN1 activates a transient epithelial-mesenchymal transition in the epithelial cells at the migrating front and drives wound closure. Once the wound is healed, these epithelial cells and their progeny can resume their original epithelial phenotype. We also found that CCN1-induced E-cadherin loss is not due to transcriptional regulation but rather protein degradation due to the collapse of adherens junctions, which is contributed by beta-catenin translocation. CCN1-activated integrin-linked kinase mediates this process. Finally, our in vivo study showed that locally neutralizing CCN1 drastically impairs wound closure, whereas local injection of recombinant CCN1 protein induces expression of vimentin and smooth muscle alpha-actin in normal gastric mucosal epithelial cells and accelerates re-epithelialization during ulcer healing. In conclusion, our study indicates that CCN1 can induce reversible epithelial-mesenchymal transition, and this feature may have great value for clinical wound healing.

  7. Adherence of skin bacteria to human epithelial cells.

    PubMed Central

    Romero-Steiner, S; Witek, T; Balish, E

    1990-01-01

    Aerobic and anaerobic bacteria isolated from human axillae were tested for their capacity to adhere to buccal epithelial cells, immortalized human epithelial (HEp-2) cells, and undifferentiated and differentiated human epithelial cells. In general, both aerobic and anaerobic diphtheroids adhered better to differentiated human epithelial cells than to HEp-2 and undifferentiated human epithelial cells (P less than 0.05). Mannose, galactose, fucose, N-acetyl-D-glucosamine, and fibronectin were also assayed for their capacity to inhibit the adherence of diphtheroids to human epithelial cells. A great deal of variability was observed in the capacity of the latter compounds to inhibit the attachment of aerobic diphtheroids to undifferentiated and differentiated epithelial cells. Overall, mannose appeared to be best at inhibiting the adherence of the aerobic diphtheroids to undifferentiated human epithelial cells. Galactose, fucose, N-acetyl-D-glucosamine, and fibronectin showed a greater capacity to inhibit attachment of aerobic diphtheroids to differentiated than to undifferentiated human epithelial cells. The inhibition of adherence to differentiated human epithelial cells varied with the microorganism and the compound tested; however, the highest and most consistent inhibition of adherence (76.1 to 88.6%) was observed with a 5% solution of N-acetyl-D-glucosamine. The in vitro adherence and adherence inhibition assays presented here demonstrate that a number of adhesins and receptors are involved in the adherence of skin bacteria to human epithelial cells and receptors on human epithelial cells are apparently altered during differentiation. PMID:2298877

  8. Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting

    PubMed Central

    Lowell, Clifford A.

    2017-01-01

    In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 × 106 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes. PMID:28180137

  9. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells.

    PubMed

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W Michael; Das, Mita

    2014-04-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.

  10. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells

    PubMed Central

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W. Michael

    2014-01-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases. PMID:24500281

  11. Adhesion and invasion of Streptococcus pneumoniae to primary and secondary respiratory epithelial cells

    PubMed Central

    Novick, Sara; Shagan, Marilous; Blau, Karin; Lifshitz, Sarit; Givon-Lavi, Noga; Grossman, Nili; Bodner, Lipa; Dagan, Ron; Nebenzahl, Yaffa Mizrachi

    2016-01-01

    The interaction between Streptococcus pneumoniae (S. pneumoniae) and the mucosal epithelial cells of its host is a prerequisite for pneumococcal disease development, yet the specificity of this interaction between different respiratory cells is not fully understood. In the present study, three areas were examined: i) The capability of the encapsulated S. pneumoniae serotype 3 strain (WU2) to adhere to and invade primary nasal-derived epithelial cells in comparison to primary oral-derived epithelial cells, A549 adenocarcinoma cells and BEAS-2B viral transformed bronchial cells; ii) the capability of the unencapsulated 3.8DW strain (a WU2 derivative) to adhere to and invade the same cells over time; and iii) the ability of various genetically-unrelated encapsulated and unencapsulated S. pneumoniae strains to adhere to and invade A549 lung epithelial cells. The results of the present study demonstrated that the encapsulated WU2 strain adhesion to and invasion of primary nasal epithelial cells was greatest, followed by BEAS-2B, A549 and primary oral epithelial cells. By contrast, the unencapsulated 3.8-DW strain invaded oral epithelial cells significantly more efficiently when compared to the nasal epithelial cells. In addition, unencapsulated S. pneumoniae strains adhered to and invaded the A459 cells significantly more efficiently than the encapsulated strains; this is consistent with previously published data. In conclusion, the findings presented in the current study indicated that the adhesion and invasion of the WU2 strain to primary nasal epithelial cells was more efficient compared with the other cultured respiratory epithelial cells tested, which corresponds to the natural course of S. pneumoniae infection and disease development. The target cell preference of unencapsulated strains was different from that of the encapsulated strains, which may be due to the exposure of cell wall proteins. PMID:27922699

  12. Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment.

    PubMed

    Yonemura, Shigenobu

    2014-01-01

    Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.

  13. Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

    PubMed

    Föllmann, W; Weber, S; Birkner, S

    2000-10-01

    Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon

  14. Ultraviolet Irradiation-Induced Volume Alteration of Corneal Epithelial Cells

    PubMed Central

    Wang, Ling; Lu, Luo

    2016-01-01

    Purpose The purpose of the study is to understand how extracellular stresses, such as ultraviolet (UV) irradiation, affect corneal epithelial cells. Cell volume changes, damage to corneal epithelial integrity, and cellular responses were assessed after exposure to UVC stresses. Methods Primary human and rabbit corneal epithelial cells were exposed to UVC light in culture conditions. Ultraviolet C irradiation–induced changes in cell size and volume were measured by real-time microscopy and self-quenching of the fluorescent dye calcein, respectively. The effects of UVC irradiation on Src and focal adhesion kinase (FAK) phosphorylation and FAK-dependent integrin signaling were detected by ELISA, immunoblotting, and immunostaining. Results Ultraviolet C irradiation induced both size and volume shifts in human and rabbit corneal epithelial cells. Ultraviolet C irradiation-induced decrease of cell volume elicited activation of Src and FAK, characterized by increased phosphorylations of SrcY416, FAKY397, and FAKY925. In addition, immunostaining studies showed UVC irradiation–induced increases in phosphorylation of FAK and formation of integrin β5 clustering. Application of Kv channel blockers, including 4-aminopyridine (4-AP), α-DTX, and depressing substance-1 (BDS-1), effectively suppressed UVC irradiation–induced cell volume changes, and subsequently inhibited UVC irradiation–induced phosphorylation of Src/FAK, and formation of integrin β5 clustering, suggesting UVC irradiation–induced volume changes and Src/FAK activation. Hyperosmotic pressure–induced volume decreases were measured in comparison with effects of UVC irradiation on volume and Src/FAK activation. However, Kv channel blocker, 4-AP, had no effect on hyperosmotic pressure–induced responses. Conclusions The present study demonstrates that UVC irradiation–induced decreases in cell volume lead to Src/FAK activation due to a rapid loss of K ions through membrane Kv channels. PMID:27978555

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

    NASA Astrophysics Data System (ADS)

    Lewis, Katherine Jean Reeder

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

  16. Isolation by Size of Epithelial Tumor Cells

    PubMed Central

    Vona, Giovanna; Sabile, Abdelmajid; Louha, Malek; Sitruk, Veronique; Romana, Serge; Schütze, Karin; Capron, Frédérique; Franco, Dominique; Pazzagli, Mario; Vekemans, Michel; Lacour, Bernard; Bréchot, Christian; Paterlini-Bréchot, Patrizia

    2000-01-01

    We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine. PMID:10623654

  17. Force mapping in epithelial cell migration

    PubMed Central

    du Roure, Olivia; Saez, Alexandre; Buguin, Axel; Austin, Robert H.; Chavrier, Philippe; Siberzan, Pascal; Ladoux, Benoit

    2005-01-01

    We measure dynamic traction forces exerted by epithelial cells on a substrate. The force sensor is a high-density array of elastomeric microfabricated pillars that support the cells. Traction forces induced by cell migration are deduced from the measurement of the bending of these pillars and are correlated with actin localization by fluorescence microscopy. We use a multiple-particle tracking method to estimate the mechanical activity of cells in real time with a high-spatial resolution (down to 2 μm) imposed by the periodicity of the post array. For these experiments, we use differentiated Madin-Darby canine kidney (MDCK) epithelial cells. Our data provide definite information on mechanical forces exerted by a cellular assembly. The maximum intensity of the forces is localized on the edge of the epithelia. Hepatocyte growth factor promotes cell motility and induces strong scattering activity of MDCK cells. Thus, we compare forces generated by MDCK cells in subconfluent epithelia versus isolated cells after hepatocyte growth factor treatment. Maximal-traction stresses at the edge of a monolayer correspond to higher values than those measured for a single cell and may be due to a collective behavior. PMID:15695588

  18. Spraying Respiratory Epithelial Cells to Coat Tissue-Engineered Constructs

    PubMed Central

    Thiebes, Anja Lena; Albers, Stefanie; Klopsch, Christian; Jockenhoevel, Stefan; Cornelissen, Christian G.

    2015-01-01

    Abstract Applying cells in a spray can overcome current hurdles in coating tissue engineered constructs with a thin layer of endo- or epithelial cells. We report here a structured study on the influences of spray application with a medical spray device on vascular smooth muscle cells (vSMCs) and respiratory epithelial cells (RECs) with and without fibrin gel. Next to viability and cytotoxicity assays, the in vitro differentiation capacity after spray processing was analyzed. For vSMC, no influence of air pressures till 0.8 bar could be shown, whereas the viability decreased for higher pressures. The viability of RECs was reduced to 88.5% with 0.4 bar air pressure. Lactate dehydrogenase-levels in the culture medium increased the first day after spraying but normalized afterward. In the short term, no differences by means of morphology and expression-specific markers for vSMCs and RECs were seen between the control and study group. In addition, in a long-term study for 28 days with the air–liquid interface, RECs differentiated and built up an organized epithelial layer with ciliary development that was comparable to the control for cells sprayed without fibrin gel. When spraying within fibrin gel, ciliary development was lower at 28 days. Thus, spraying of vSMCs and RECs was proved to be a suitable method for tissue engineering. Especially for RECs, this application is of special significance when coating luminal structures or other unfavorable topographies. PMID:26309803

  19. Lactobacillus acidophilus contributes to a healthy environment for vaginal epithelial cells.

    PubMed

    Pi, Woojin; Ryu, Jae-Sook; Roh, Jaesook

    2011-09-01

    Lactobacillus species in the female genital tract are thought to act as a barrier to infection. Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. acidophilus and the acidity of the culture medium were measured. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state. L. acidophilus showed exponential growth for the first 6 hr until 9 hr, and the pH was maintained close to 4.0-5.0 at 24 hr after culture, consistent with previous studies. The growth curve of L. acidophilus or the pH values were relatively unaffected by co-culture with MS74 cells, confirming that L. acidophilus maintains a low pH in the presence of MS74 cells. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. On the other hand, MS74 cells co-cultured with L. acidophilus more firmly attached to the culture plate, and a higher number of cells were present compared to cells cultured in the absence of L. acidophilus. These results indicate that L. acidophilus increases MS74 cell proliferation and viability, suggesting that lactobacilli may contribute to the healthy environment for vaginal epithelial cells.

  20. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells.

    PubMed Central

    Simon, R H; DeHart, P D; Todd, R F

    1986-01-01

    The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing. Images PMID:3771800

  1. Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway.

    PubMed

    Said, Hamid M; Wang, Shuling; Ma, Thomas Y

    2005-07-15

    In mammalian cells (including those of the ocular system), the water-soluble vitamin B2 (riboflavin, RF) assumes an essential role in a variety of metabolic reactions and is critical for normal cellular functions, growth and development. Cells of the human retinal pigment epithelium (hRPE) play an important role in providing a sufficient supply of RF to the retina, but nothing is known about the mechanism of the vitamin uptake by these cells and its regulation. Our aim in the present study was to address this issue using the hRPE ARPE-19 cells as the retinal epithelial model. Our results show RF uptake in the hRPE to be: (1) energy and temperature dependent and occurring without metabolic alteration in the transported substrate, (2) pH but not Na+ dependent, (3) saturable as a function of concentration with an apparent Km of 80 +/- 14 nM, (4) trans-stimulated by unlabelled RF and its structural analogue lumiflavine, (5) cis-inhibited by the RF structural analogues lumiflavine and lumichrome but not by unrelated compounds, and (6) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) as well as by the Na+ -H+ exchange inhibitor amiloride and the sulfhydryl group inhibitor p-chloromercuriphenylsulphonate (p-CMPS). Maintaining the hRPE cells in a RF-deficient medium led to a specific and significant up-regulation in RF uptake which was mediated via changes in the number and affinity of the RF uptake carriers. While modulating the activities of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, protein tyrosine kinase (PTK)-, and nitric oxide (NO)-mediated pathways were found to have no role in regulating RF uptake, a role for the Ca2+ -calmodulin-mediated pathway was observed. These studies demonstrate for the first time the involvement of a specialized carrier-mediated mechanism for RF uptake by hRPE cells and show that the process is

  2. Involvement of Egr-1 in lens epithelial cell death induced by selenite.

    PubMed

    Nakajima, T; Belusko, P B; Walkup, R D; Azuma, M; Shearer, T R

    2006-05-01

    Selenite-overdose cataract in young rats may be caused by an initial insult to the lens epithelial cells. Our previous DNA array analysis revealed a significant increase in the expression of mRNA for early growth response protein-1 (Egr-1) in lens epithelial cells after injection of selenite. This suggested that up-regulation of Egr-1 mRNA may be involved in lens epithelial cell death. The purpose of the present experiment was to further clarify the involvement of Egr-1 in lens epithelial cell death induced by selenite. Rat lens epithelial explants were cultured with sodium selenite. Selenite caused epithelial explants to leak LDH into the medium. During LDH leakage, increased expression of mRNA for Egr-1 was observed by RT-PCR. To further test the involvement of Egr-1 in selenite-induced cell death, mouse lens epithelial cell line (alpha-TN4 cells) was treated with antisense oligonucleotide for Egr-1. Antisense oligonucleotide for Egr-1 significantly diminished expression of Egr-1 protein and leakage of LDH. These results suggested that increased activity of Egr-1 may be a factor in lens epithelial cell death induced by selenite.

  3. Roles of Wnt/{beta}-catenin signaling in epithelial differentiation of mesenchymal stem cells

    SciTech Connect

    Wang, Yajing; Sun, Zhaorui; Qiu, Xuefeng; Li, Yan; Qin, Jizheng; Han, Xiaodong

    2009-12-25

    Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/{beta}-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/{beta}-catenin signaling were determined, suggested down-regulation of Wnt/{beta}-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3{alpha} can inhibit the epithelial differentiation of MSCs. A loss of {beta}-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated {beta}-catenin expression and subsequently decreased {beta}-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/{beta}-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.

  4. Efficient cultivation conditions for human limbal epithelial cells.

    PubMed

    Kim, Mee Kum; Lee, Jae Lim; Oh, Joo Youn; Shin, Mi Sun; Shin, Kyeong Seon; Wee, Won Ryang; Lee, Jin Hak; Park, Ki Sook; Son, Young Sook

    2008-10-01

    To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

  5. Serratia marcescens is injurious to intestinal epithelial cells

    PubMed Central

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

    2014-01-01

    Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens. PMID:25426769

  6. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation.

    PubMed

    Taylor, A W; Dixit, S; Yu, J

    2015-01-29

    The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In

  7. Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

    PubMed Central

    Huang, Haiyan; Xia, Bo; Liu, Hongya; Li, Jie; Lin, Shaolin; Li, Tiyuan; Liu, Jianjun; Li, Hui

    2015-01-01

    Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery. PMID:25861018

  8. Stromal–Epithelial Crosstalk Provides a Suitable Microenvironment for the Progression of Ovarian Cancer Cells in Vitro

    PubMed Central

    Fu, Shilong; Dong, Lihua; Sun, Wei; Xu, Yi; Gao, Li; Miao, Yi

    2013-01-01

    The tumor microenvironment plays an important role in the progression of cancer. This study focused on carcinoma-associated fibroblasts (CAFs) and stromal–epithelial interaction between CAFs and epithelial ovarian carcinoma (EOC) cells. We isolated and established primary cultures of CAFs and co-cultured CAFs and EOC cells in vitro. The co-culture conditioned medium (CC-CM) was harvested and its influence on EOC cells was examined. Cytokine, chemokine, and growth factor levels were screened using a biotin label-based human antibody array system. We found that the stromal–epithelial crosstalk provided a suitable microenvironment for the progression of ovarian cancer cells in vitro. PMID:24147897

  9. Azithromycin kills invasive Aggregatibacter actinomycetemcomitans in gingival epithelial cells.

    PubMed

    Lai, Pin-Chuang; Walters, John D

    2013-03-01

    Aggregatibacter actinomycetemcomitans invades periodontal pocket epithelium and is therefore difficult to eliminate by periodontal scaling and root planing. It is susceptible to azithromycin, which is taken up by many types of mammalian cells. This led us to hypothesize that azithromycin accumulation by gingival epithelium could enhance the killing of intraepithelial A. actinomycetemcomitans. [(3)H]azithromycin transport by Smulow-Glickman gingival epithelial cells and SCC-25 oral epithelial cells was characterized. To test our hypothesis, we infected cultured Smulow-Glickman cell monolayers with A. actinomycetemcomitans (Y4 or SUNY 465 strain) for 2 h, treated them with gentamicin to eliminate extracellular bacteria, and then incubated them with azithromycin for 1 to 4 h. Viable intracellular bacteria were released, plated, and enumerated. Azithromycin transport by both cell lines exhibited Michaelis-Menten kinetics and was competitively inhibited by l-carnitine and several other organic cations. Cell incubation in medium containing 5 μg/ml azithromycin yielded steady-state intracellular concentrations of 144 μg/ml in SCC-25 cells and 118 μg/ml in Smulow-Glickman cells. Azithromycin induced dose- and time-dependent intraepithelial killing of both A. actinomycetemcomitans strains. Treatment of infected Smulow-Glickman cells with 0.125 μg/ml azithromycin killed approximately 29% of the intraepithelial CFU of both strains within 4 h, while treatment with 8 μg/ml azithromycin killed ≥82% of the CFU of both strains (P < 0.05). Addition of carnitine inhibited the killing of intracellular bacteria by azithromycin (P < 0.05). Thus, human gingival epithelial cells actively accumulate azithromycin through a transport system that facilitates the killing of intraepithelial A. actinomycetemcomitans and is shared with organic cations.

  10. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2010-10-01

    breast is highly expressed by luminal epithelial cells and is less expressed by basal cells19,20. In contrast, CD49f (a6 integrin) has an inverse pattern...mouse stretched on its back. The hose and nose cone from the anesthetic vaporizer are securely attached to one side of the plate, and a heated pad is...the mouse by a nose cone. Check that the mouse has reached surgical anesthesia by loss of pedal withdrawal reflex . ! cautIon Institutional review

  11. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

    Gharib, Sina A.; Pippin, Jeffrey W.; Ohse, Takamoto; Pickering, Scott G.; Krofft, Ronald D.; Shankland, Stuart J.

    2014-01-01

    Very little is known about the function of glomerular parietal epithelial cells (PECs). In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire. PMID:25127402

  12. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans.

  13. A dynamic podosome-like structure of epithelial cells.

    PubMed

    Spinardi, Laura; Rietdorf, Jens; Nitsch, Lucio; Bono, Maria; Tacchetti, Carlo; Way, Michael; Marchisio, Pier Carlo

    2004-05-01

    Focal contacts and hemidesmosomes are cell-matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin, and zyxin. We also show that the maintenance of the actin core and hemidesmosomes is dependent on actin polymerization, src-family kinases, and Grb2, but not on microtubules. Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of beta4 integrin subunit to the actin core before its positioning at hemidesmosomes. When 804G cells are induced to migrate, actin cores as well as hemidesmosomes disappear and beta4 integrin subunit becomes co-localized with dynamic actin at leading edges. We show that podosome-like structures are not unique to cells of mesenchymal origin, but also appear in epithelial cells, where they seem to be related to basement membrane adhesion.

  14. SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture.

    PubMed

    Velásquez, Juan C; Hidalgo, Alejandro A; Villagra, Nicolás; Santiviago, Carlos A; Mora, Guido C; Fuentes, Juan A

    2016-08-01

    The genomic island 9 (SPI-9) from Salmonella enterica serovar Typhi (S. Typhi) carries three ORFs (STY2876, STY2877, STY2878) presenting 98 % identity with a type 1 secretory apparatus (T1SS), and a single ORF (STY2875) similar to a large RTX-like protein exhibiting repeated Ig domains. BapA, the Salmonella enterica serovar Enteritidis orthologous to S. Typhi STY2875, has been associated with biofilm formation, and is described as a virulence factor in mice. Preliminary in silico analyses revealed that S. Typhi STY2875 ORF has a 600 bp deletion compared with S. Enteritidis bapA, suggesting that S. Typhi STY2875 might be non-functional. At present, SPI-9 has not been studied in S. Typhi. We found that the genes constituting SPI-9 are arranged in an operon whose promoter was up-regulated in high osmolarity and low pH in a RpoS-dependent manner. All the proteins encoded by S. Typhi SPI-9 were located at the membrane fraction, consistent with their putative role as T1SS. Furthermore, SPI-9 contributed to adherence of S. Typhi to epithelial cells when bacteria were grown under high osmolarity or low pH. Under the test conditions, S. Typhi SPI-9 did not participate in biofilm formation. SPI-9 is functional in S. Typhi and encodes an adhesin induced under conditions normally found in the intestine, such as high osmolarity. Hence, this is an example of a locus that might be designated a pseudogene by computational approaches but not by direct biological assays.

  15. Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells.

    PubMed Central

    Corbeil, L B; Hodgson, J L; Jones, D W; Corbeil, R R; Widders, P R; Stephens, L R

    1989-01-01

    Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective. Images PMID:2471692

  16. Mediation of cimetidine secretion by P-glycoprotein and a novel H(+)-coupled mechanism in cultured renal epithelial monolayers of LLC-PK1 cells.

    PubMed Central

    Dudley, A. J.; Brown, C. D.

    1996-01-01

    1. Previous studies have shown that the weak base, cimetidine, is actively secreted by the renal proximal tubule. In this study we have examined the transport of cimetidine by renal LLC-PK1 epithelial cell monolayers. 2. In LLC-PK1 cell monolayers the basal-to-apical flux of cimetidine was significantly greater than the apical-to basal flux, consistent with net secretion of cimetidine in a basal-to-apical direction. 3. Net secretion of cimetidine was significantly (70%) reduced by the addition of either 100 microM verapamil or 100 microM nifedipine to the apical membrane. The reduction in net secretion was the result of an inhibition of basal-to-apical flux; these agents had no effect upon flux in the apical-to-basal direction. These results suggest that cimetidine secretion is mediated primarily by P-glycoprotein located in the apical membrane. In addition we found no evidence of a role for organic cation antiport in the secretion of cimetidine. 4. In the presence of an inwardly directed proton gradient across the apical membrane (pH 6.0), cimetidine secretion was significantly reduced compared to that measured at an apical pH of 7.4. The reduction in net secretion at pH 6.0 was the result of a stimulation of cimetidine uptake across the apical membrane. This pH-dependent uptake mechanism was sensitive to inhibition by DIDS (100 microM). 5. Experiments with BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) loaded monolayers demonstrated that cimetidine influx across the apical membrane was associated with proton flow into the cell and was sensitive to inhibition by DIDS. 6. These results suggest that net secretion of cimetidine across the apical membrane is a function of the relative magnitudes of cimetidine secretion mediated by P-glycoprotein and cimetidine absorption mediated by a novel proton-coupled, DIDS-sensitive transport mechanism. PMID:8882608

  17. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  18. Intestinal epithelial culture under an air-liquid interface: a tool for studying human and mouse esophagi.

    PubMed

    Yokobori, T; Suzuki, S; Miyazaki, T; Sohda, M; Sakai, M; Tanaka, N; Ozawa, D; Hara, K; Honjo, H; Altan, B; Fukuchi, M; Ishii, H; Iwatsuki, M; Sugimachi, K; Sudo, T; Iwaya, T; Nishida, N; Mimori, K; Kuwano, H; Mori, M

    2016-10-01

    This study investigated whether an intestinal epithelial culture method can be applied to mouse and human esophageal cultures. The esophagi harvested from 1-day-old mice and adult humans were maintained in collagen gels. A commercially available culture medium for human embryonic stem cells was used for the human esophageal culture. We discovered that the intestinal epithelial culture method can be successfully applied to both mouse and human esophageal cultures. The long-term cultured esophageal organoids were rod-like luminal structures lined with myofibroblasts. We discovered that regeneration of the esophageal mucosal surface can be almost completely achieved in vitro, and the advantage of this method is that organoid cultures may be generated using host-derived fibroblasts as a niche. This method is a promising tool for mouse and human research in intestinal biology, carcinogenesis, and regenerative medicine.

  19. Bronchial epithelial cells release monocyte chemotactic activity in response to smoke and endotoxin

    SciTech Connect

    Koyama, S.; Rennard, S.I.; Leikauf, G.D.; Robbins, R.A. )

    1991-08-01

    An increase in mononuclear phagocytes occurs within the airways during airway inflammation. Bronchial epithelial cells could release monocyte chemotactic activity and contribute to this increase. To test this hypothesis, bovine bronchial epithelial cells were isolated and maintained in culture. Bronchial epithelial cell culture supernatant fluids were evaluated for monocyte chemotactic activity. Epithelial cell culture supernatant fluids attracted significantly greater numbers of monocytes compared to media alone and the number of monocytes attracted increased in a time dependent manner. Endotoxin and smoke extract induced a dose and time dependent release of monocyte chemotactic activity compared with cells cultured in media (52.5 {plus minus} 2.6 (endotoxin), 30.5 {plus minus} 2.3 (smoke) vs 20.5 {plus minus} 2.2 cells/high power field (HPF) p less than 0.001). The released activity was chemotactic by checkerboard analysis. Stimulation of the epithelial cells by opsonized zymosan, calcium ionophore (A23187), and PMA also resulted in an increase in monocyte chemotactic activity (p less than 0.01). Because the release of activity was blocked by the lipoxygenase inhibitors, nordihydroguaiaretic acid and diethycarbamazine, epithelial cell monolayers were cultured with 3 microCi (3H)arachidonic acid for 24 h and then exposed to A23187, PMA, or both stimuli, for 4, 8, and 24 h. Analysis of the released 3H activity was performed with reverse-phase HPLC and revealed that the major lipoxygenase product was leukotriene B4. These data suggest that monocytes may be recruited into airways in response to chemotactic factors released by bronchial epithelial cells.

  20. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

    SciTech Connect

    Jin, Hua; Shen, Shuijie; Chen, Xiaoyan; Zhong, Dafang; Zheng, Jiang

    2012-06-15

    Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.

  1. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

    PubMed

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection.

  2. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses

    PubMed Central

    Ozbun, Michelle A.; Patterson, Nicole A.

    2014-01-01

    Papillomaviruses have a strict tropism for epithelial cells and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro wherein virion morphogenesis occurs under cooperative viral and cellular cues requires the cultivation of epithelium. Presented in the first section of this unit is a protocol for growing differentiating epithelial tissues, whose structure and function mimics many important morphological and biochemical aspects of normal skin. The technique, pioneered by Asslineau and Pruniéras (Asselineau and Prunieras 1984) and modified by Kopan et al. (Kopan et al. 1987), involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname “raft” cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, as well as keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single step virus growth

  3. Density dependent polarized secretion of a prostatic epithelial cell line.

    PubMed

    Djakiew, D; Pflug, B; Delsite, R; Lynch, J H; Onoda, M

    1992-01-01

    The polarized secretions (apical/basal) of newly synthesized total protein and proteases from prostatic epithelial sheets of PA-III cells grown in dual compartment chambers were investigated at various cell densities and culture conditions. PA-III cells grown in a serum free defined medium (SFDM) form morphologically polarized monolayers of epithelial cells. These cells secreted their 35S-methionine labeled total protein in a predominantly apical direction (apical/basal ratio, 4-8 fold), with a lesser proportion of protein secreted apically at lower cell densities of the PA-III cell monolayer. PA-III cells grown in 5% fetal calf serum (FCS) are morphologically squamous, comparable to the anaplastic phenotype, and exhibited an inversion of polarized total protein secretion (apical/basal ratio, 0.4-0.9 fold), with an increased proportion of total protein secreted in a basal direction at lower cell densities. Since the culture of PA-III cells in FCS may approximate the anaplastic phenotype we investigated the polarized secretion of proteases from these cells at various cell densities, and compared them with the secretory pattern of protease secretion from polarized PA-III cells cultured in SFDM. At lower cell densities of the PA-III cells grown in FCS the polarity of protease secretion was inverted such that metalloproteinases, tissue type plasminogen activator, and a 72 kD gelatinase were secreted in a predominantly basal direction, as well as urokinase and a gelatinase of 26 kD that were secreted more or less equally into the apical and basal compartments of the chambers. On the other hand, for cultures of PA-III cells grown in SFDM the aforementioned proteases exhibited predominantly an apically directed polarity of secretion. These results suggest that the anaplastic phenotype characterized by a loss of polarized structure may also be characterized by a functional loss or inversion of polarized secretion. The consequences of such a loss or inversion of polarized

  4. Bim Regulation of Lumen Formation in Cultured Mammary Epithelial Acini Is Targeted by Oncogenes

    PubMed Central

    Reginato, Mauricio J.; Mills, Kenna R.; Becker, Esther B. E.; Lynch, Danielle K.; Bonni, Azad; Muthuswamy, Senthil K.; Brugge, Joan S.

    2005-01-01

    Epithelial cells organize into cyst-like structures that contain a spherical monolayer of cells that enclose a central lumen. Using a three-dimensional basement membrane culture model in which mammary epithelial cells form hollow, acinus-like structures, we previously demonstrated that lumen formation is achieved, in part, through apoptosis of centrally localized cells. We demonstrate that the proapoptotic protein Bim may selectively trigger apoptosis of the centrally localized acinar cells, leading to temporally controlled lumen formation. Bim is not detectable during early stages of three-dimensional mammary acinar morphogenesis and is then highly upregulated in all cells of acini, coincident with detection of apoptosis in the centrally localized acinar cells. Inhibition of Bim expression by RNA interference transiently blocks luminal apoptosis and delays lumen formation. Oncogenes that induce acinar luminal filling, such as ErbB2 and v-Src, suppress expression of Bim through a pathway dependent on Erk-mitogen-activated protein kinase; however, HPV 16 E7, an oncogene that stimulates cell proliferation but not luminal filling, is unable to reduce Bim expression. Thus, Bim is a critical regulator of luminal apoptosis during mammary acinar morphogenesis in vitro and may be an important target of oncogenes that disrupt glandular epithelial architecture. PMID:15899862

  5. Higher level of replication efficiency of 2009 (H1N1) pandemic influenza virus than those of seasonal and avian strains: kinetics from epithelial cell culture and computational modeling.

    PubMed

    Mitchell, Hugh; Levin, Drew; Forrest, Stephanie; Beauchemin, Catherine A A; Tipper, Jennifer; Knight, Jennifer; Donart, Nathaniel; Layton, R Colby; Pyles, John; Gao, Peng; Harrod, Kevin S; Perelson, Alan S; Koster, Frederick

    2011-01-01

    The pathogenicity and transmission of influenza A viruses are likely determined in part by replication efficiency in human cells, which is the net effect of complex virus-host interactions. H5N1 avian, H1N1 seasonal, and H1N1 2009 pandemic influenza virus strains were compared by infecting human differentiated bronchial epithelial cells in air-liquid interface cultures at relatively low virus particle/cell ratios. Differential equation and computational models were used to characterize the in vitro kinetic behaviors of the three strains. The models were calibrated by fitting experimental data in order to estimate difficult-to-measure parameters. Both models found marked differences in the relative values of p, the virion production rate per cell, and R(0), an index of the spread of infection through the monolayer, with the values for the strains in the following rank order (from greatest to least): pandemic strain, followed by seasonal strain, followed by avian strain, as expected. In the differential equation model, which treats virus and cell populations as well mixed, R(0) and p varied proportionately for all 3 strains, consistent with a primary role for productivity. In the spatially explicit computational model, R(0) and p also varied proportionately except that R(0) derived for the pandemic strain was reduced, consistent with constrained viral spread imposed by multiple host defenses, including mucus and paracrine antiviral effects. This synergistic experimental-computational strategy provides relevant parameters for identifying and phenotyping potential pandemic strains.

  6. Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium.

    PubMed

    Nakanuma, Y; Katayanagi, K; Kawamura, Y; Yoshida, K

    1997-10-01

    Several models for preparing and isolating human and animal gallbladder epithelial cells, including low-grade gallbladder carcinoma cells, as well as proposed systems for culturing these isolated epithelial cells are reviewed here. Several reports concerning tissue culture of the gallbladder are also reviewed. The cell culture systems are divided into monolayer cell culture on collagen-coated or uncoated culture dishes or other culture substrate and three-dimensional cell culture in collagen gel. To prepare and isolate gallbladder epithelial cells, digestion of the gallbladder mucosa, abrasion of the mucosal epithelial cells, and excision of epithelial outgrowth of mucosal explants are applied. In monolayer cell culture, most of the specific biological features of isolated and cultured cells characteristic to the gallbladder are gradually lost after several passages, though quantitative and objective analyses of the pathophysiology of cultured cells and their secretory substances can be performed. Tissue culture using explants of the gallbladder has mainly been used for physiological studies of the gallbladder, such as investigating the transport of water and electrolytes. In this tissue culture system, quantitative assessment is difficult, though the original and specific biological and histological characteristics of the gallbladder are retained. Three-dimensional collagen gel culture could be an ideal model combining monolayer cell culture and tissue culture systems, and create controllable conditions or environments when several biologically active substances, such as growth factors, proinflammatory cytokines and adhesion molecules, are added to the culture medium. Advantages and shortcomings of individual cultivation models are discussed, and selecting the culture model most appropriate to the purpose of the study will facilitate investigations of the biology and pathogenetic mechanisms of gallbladder diseases such as cholelithiasis.

  7. Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR

    PubMed Central

    Patil, Pratima U.; D'Ambrosio, Julia; Inge, Landon J.; Mason, Robert W.; Rajasekaran, Ayyappan K.

    2015-01-01

    ABSTRACT In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial–mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells. PMID:26483386

  8. Release of simian virus 40 virions from epithelial cells is polarized and occurs without cell lysis.

    PubMed Central

    Clayson, E T; Brando, L V; Compans, R W

    1989-01-01

    We have investigated the process of release of simian virus 40 (SV40) virions from several monkey kidney cell lines. High levels of virus release were observed prior to any significantly cytopathic effects in all cell lines examined, indicating that SV40 utilizes a mechanism for escape from the host cell which does not involve cell lysis. We demonstrate that SV40 release was polarized in two epithelial cell types (Vero C1008 and primary African green monkey kidney cells) grown on permeable supports; release of virus occurs almost exclusively at apical surfaces. In contrast, equivalent amounts of SV40 virions were recovered from apical and basal culture fluids of nonpolarized CV-1 cells. SV40 virions were observed in large numbers on apical surfaces of epithelial cells and in cytoplasmic smooth membrane vesicles. The sodium ionophore monensin, an inhibitor of vesicular transport, was found to inhibit SV40 release without altering viral protein synthesis or infectious virus production. Images PMID:2539518

  9. Macrophages regulate expression of α1,2-fucosyltransferase genes in human endometrial epithelial cells.

    PubMed

    Nakamura, Hitomi; Jasper, Melinda J; Hull, M Louise; Aplin, John D; Robertson, Sarah A

    2012-04-01

    The epithelial cell surface of the endometrium undergoes substantial biochemical changes to allow embryo attachment and implantation in early pregnancy. We hypothesized that tissue macrophages influence these events to promote uterine receptivity. To investigate the role of macrophages in regulating epithelial cell expression of genes linked to glycan-mediated embryo adhesion, Ishikawa, RL95-2 and HEC1A endometrial epithelial cells were cultured alone or with unactivated or lipopolysaccharide-activated monocytic U937 cells, separated using transwell inserts. Expression of mRNAs encoding two α1,2-fucosyltransferases (FUT1, FUT2) was increased in all three epithelial cell lines following co-culture with U937 cells, and was associated with increased fucosylation of cell surface glycoproteins detected using lectins from Ulex europaeus (UEA-1) and Dolichos biflorus (DBA). FUT1 induction by U937 cells also occurred in primary endometrial epithelial cells collected in luteal but not proliferative phase. Activation of the interleukin-6 (IL6)/leukemia inhibitory factor (LIF) cytokine signaling pathway with phosphorylation of STAT3 and elevated SOCS3 mRNA expression was evident in epithelial cells stimulated by U937 co-culture. Several recombinant macrophage-secreted cytokines exerted stimulatory or inhibitory effects on FUT1 and FUT2 mRNA expression, and the macrophage-derived cytokine LIF partially replicated the effects of U937 cells on both FUT1 and FUT2 expression and UEA-1 and DBA lectin reactivity in Ishikawa cells. These results suggest that macrophage-derived factors including LIF might facilitate development of an implantation-receptive endometrium by regulating surface glycan structures in epithelial cells. Abnormal phenotypes or altered abundance of uterine macrophages could contribute to the pathophysiology of primary unexplained infertility in women.

  10. Polar release of pathogenic Old World hantaviruses from renal tubular epithelial cells

    PubMed Central

    2012-01-01

    Background Epithelio- and endotheliotropic viruses often exert polarized entry and release that may be responsible for viral spread and dissemination. Hantaviruses, mostly rodent-borne members of the Bunyaviridae family infect epithelial and endothelial cells of different organs leading to organ dysfunction or even failure. Endothelial and renal epithelial cells belong to the target cells of Old World hantavirus. Therefore, we examined the release of hantaviruses in several renal epithelial cell culture models. We used Vero cells that are commonly used in hantavirus studies and primary human renal epithelial cells (HREpC). In addition, we analyzed MDCKII cells, an epithelial cell line of a dog kidney, which represents a widely accepted in vitro model of polarized monolayers for their permissiveness for hantavirus infection. Results Vero C1008 and primary HREpCs were grown on porous-support filter inserts for polarization. Monolayers were infected with hantavirus Hantaan (HTNV) and Puumala (PUUV) virus. Supernatants from the apical and basolateral chamber of infected cells were analyzed for the presence of infectious particles by re-infection of Vero cells. Viral antigen and infectious particles of HTNV and PUUV were exclusively detected in supernatants collected from the apical chamber of infected Vero C1008 cells and HREpCs. MDCKII cells were permissive for hantavirus infection and polarized MDCKII cells released infectious hantaviral particles from the apical surface corresponding to the results of Vero and primary human epithelial cells. Conclusions Pathogenic Old World hantaviruses are released from the apical surface of different polarized renal epithelial cells. We characterized MDCKII cells as a suitable polarized cell culture model for hantavirus infection studies. PMID:23194647

  11. Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

    SciTech Connect

    Twite, Nicolas; Andrei, Graciela; Kummert, Caroline; Donner, Catherine; Perez-Morga, David; De Vos, Rita; Snoeck, Robert; Marchant, Arnaud

    2014-07-15

    Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMV by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.

  12. Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse.

    PubMed

    Yamben, Idella F; Rachel, Rivka A; Shatadal, Shalini; Copeland, Neal G; Jenkins, Nancy A; Warming, Soren; Griep, Anne E

    2013-12-01

    The integrity and function of epithelial tissues depend on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling.

  13. Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells.

    PubMed

    Mendicino, J; Sangadala, S

    1999-11-01

    The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by simian virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines. The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of 35SO4 were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons. These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.

  14. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2009-10-01

    Appendix……………………………………………………………………………… 11 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A method for... Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability...Abstracts Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turshvili, Sam Aparicio , Joanne Emerman and Connie Eaves, “Identification of Human Mammary

  15. Proteomic Analysis of Nasal Epithelial Cells from Cystic Fibrosis Patients

    PubMed Central

    Papon, Jean-François; Chhuon, Cerina; Zadigue, Patricia; Prulière-Escabasse, Virginie; Amselem, Serge; Escudier, Estelle; Coste, André; Edelman, Aleksander

    2014-01-01

    The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology. PMID:25268127

  16. Development and limitations of lentivirus vectors as tools for tracking differentiation in prostate epithelial cells.

    PubMed

    Frame, Fiona M; Hager, Stefanie; Pellacani, Davide; Stower, Mike J; Walker, Hannah F; Burns, Julie E; Collins, Anne T; Maitland, Norman J

    2010-11-15

    To investigate hierarchy in human prostate epithelial cells, we generated recombinant lentiviruses, infected primary cultures and cell lines, and followed their fate in vitro. The lentiviruses combined constitutive promoters including CMV and β-actin, or late-stage differentiation promoters including PSCA (prostate stem cell antigen) and PSAPb (prostate specific antigen/probasin) driving expression of monomeric, dimeric and tetrameric fluorescent proteins. Significantly, rare CD133(+) cells from primary prostate epithelial cultures were successfully infected and activation of late-stage promoters was observed in basal epithelial cultures following induction of differentiation. Lentiviruses also infected CD133(+) cells within the P4E6 cell line. However, promoter silencing was observed in several cell lines (P4E6, BPH-1, PC3). We examined the promoter methylation status of the lentiviral insertions in heterogeneously fluorescent cultures from PC3 clones and found that DNA methylation was not the primary mechanism of silencing of the CMV promoter. We also describe limitations to the lentivirus system including technical challenges due to low titers and low infection efficiency in primary cultures. However, we have identified a functional late-stage promoter that indicates differentiation from a basal to a luminal phenotype and demonstrate that this strategy for lineage tracking of prostate epithelial cells is valid with further optimisation.

  17. Contact-mediated control of radial migration of corneal epithelial cells

    PubMed Central

    Walczysko, Petr; Rajnicek, Ann M.

    2016-01-01

    Purpose Patients with a heterozygous mutation in the gene encoding the transcription factor, PAX6, have a degenerative corneal opacity associated with failure of normal radial epithelial cell migration across the corneal surface and a reported wound healing defect. This study investigated the guidance mechanisms that drive the directed migration of corneal epithelial cells. Methods In vivo corneal epithelial wounding was performed in adult wild-type and Pax6+/− mice, and the healing migration rates were compared. To investigate the control of the cell migration direction, primary corneal epithelial cells from wild-type and Pax6+/− mice were plated on grooved quartz substrates, and alignment relative to the grooves was assayed. A reconstructed corneal culture system was developed in which dissociated wild-type and genetically mutant corneal epithelial cells could be cultured on a de-epithelialized corneal stroma or basement membrane and their migration assayed with time-lapse microscopy. Results The Pax6+/− cells efficiently re-epithelialized corneal wounds in vivo but had mild slowing of healing migration compared to the wild-type. Cells aligned parallel to quartz grooves in vitro, but the Pax6+/− cells were less robustly oriented than the wild-type. In the reconstructed corneal culture system, corneal epithelial cells continued to migrate radially, showing that the cells are guided by contact-mediated cues from the basement membrane. Recombining wild-type and Pax6 mutant corneal epithelial cells with wild-type and Pax6 mutant corneal stroma showed that normal Pax6 dosage was required autonomously in the epithelial cells for directed migration. Integrin-mediated attachment to the substrate, and intracellular PI3Kγ activity, were required for migration. Pharmacological inhibition of cAMP signaling randomized migration tracks in reconstructed corneas. Conclusions Striking patterns of centripetal migration of corneal epithelial cells observed in vivo are

  18. Quantification of regenerative potential in primary human mammary epithelial cells

    PubMed Central

    Linnemann, Jelena R.; Miura, Haruko; Meixner, Lisa K.; Irmler, Martin; Kloos, Uwe J.; Hirschi, Benjamin; Bartsch, Harald S.; Sass, Steffen; Beckers, Johannes; Theis, Fabian J.; Gabka, Christian; Sotlar, Karl; Scheel, Christina H.

    2015-01-01

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

  19. Quantification of regenerative potential in primary human mammary epithelial cells.

    PubMed

    Linnemann, Jelena R; Miura, Haruko; Meixner, Lisa K; Irmler, Martin; Kloos, Uwe J; Hirschi, Benjamin; Bartsch, Harald S; Sass, Steffen; Beckers, Johannes; Theis, Fabian J; Gabka, Christian; Sotlar, Karl; Scheel, Christina H

    2015-09-15

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.

  20. Trehalose maintains vitality of mouse epididymal epithelial cells and mediates gene transfer.

    PubMed

    Qu, Bin; Gu, Yihua; Shen, Jian; Qin, Jinzhou; Bao, Jianqiang; Hu, Yuan; Zeng, Wenxian; Dong, Wuzi

    2014-01-01

    In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120-240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epithelial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer.